Immunogenic compositions comprising conjugated capsular saccharide antigens, kits comprising the same and uses thereof

ABSTRACT

The present invention relates to new immunogenic compositions comprising conjugated Streptococcus pneumoniae capsular saccharide antigens (glycoconjugates), kits comprising said immunogenic compositions and uses thereof. Immunogenic compositions of the present invention will typically comprise at least one glycoconjugate from a S. pneumoniae serotype not found in PREVNAR®, SYNFLORIX® and/or PREVNAR 13®. The invention also relates to vaccination of human subjects, in particular infants and elderly, against pneumoccocal infections using said novel immunogenic compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. Ser. No.15/214,693, filed on Jul. 20, 2016, (allowed), which claims the benefitunder 35 U.S.C. § 119(e) of U.S. Provisional Application Ser. No.62/194,965, filed Jul. 21, 2015, both of which are incorporated byreference in their entirety.

REFERENCE TO SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence listing entitled “PC72217B_ST25.txt”, createdon Sep. 25, 2018 and having a size of 12 KB. The sequence listingcontained in this .txt file is part of the specification and is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to new immunogenic compositions comprisingconjugated capsular saccharide antigens (glycoconjugates), kitscomprising said immunogenic compositions and uses thereof. Immunogeniccompositions of the present invention will typically compriseglycoconjugates, wherein the saccharides are derived from serotypes ofStreptococcus pneumoniae. The invention also relates to vaccination ofhuman subjects, in particular infants and elderly, against pneumoccocalinfections using said novel immunogenic compositions and kits.

BACKGROUND OF THE INVENTION

Infections caused by pneumococci are a major cause of morbidity andmortality all over the world. Pneumonia, febrile bacteraemia andmeningitis are the most common manifestations of invasive pneumococcaldisease, whereas bacterial spread within the respiratory tract mayresult in middle-ear infection, sinusitis or recurrent bronchitis.Compared with invasive disease, the non-invasive manifestations areusually less severe, but considerably more common.

In Europe and the United States, pneumococcal pneumonia is the mostcommon community-acquired bacterial pneumonia, estimated to affectapproximately 100 per 100,000 adults each year. The correspondingfigures for febrile bacteraemia and meningitis are 15-19 per 100 000 and1-2 per 100,000, respectively. The risk for one or more of thesemanifestations is much higher in infants and elderly people, as well asimmune compromised persons of any age. Even in economically developedregions, invasive pneumococcal disease carries high mortality; foradults with pneumococcal pneumonia the mortality rate averages 10%-20%,whilst it may exceed 50% in the high-risk groups. Pneumonia is by farthe most common cause of pneumococcal death worldwide.

The etiological agent of pneumococcal diseases, Streptococcus pneumoniae(pneumococcus), is a Gram-positive encapsulated coccus, surrounded by apolysaccharide capsule. Differences in the composition of this capsulepermit serological differentiation between about 91 capsular types, someof which are frequently associated with pneumococcal disease, othersrarely. Invasive pneumococcal infections include pneumonia, meningitisand febrile bacteremia; among the common non-invasive manifestations areotitis media, sinusitis and bronchitis.

Pneumococcal conjugate vaccines (PCVs) are pneumococcal vaccines used toprotect against disease caused by S. pneumoniae (pneumococcus). Thereare currently three PCV vaccines available on the global market:PREVNAR® (PREVENAR in some countries) (heptavalent vaccine), SYNFLORIX®(a decavalent vaccine) and PREVNAR 13® (PREVENAR 13® in some countries)(tridecavalent vaccine).

The recent development of widespread microbial resistance to essentialantibiotics and the increasing number of immunocompromised personsunderline the need for pneumococcal vaccines with even broaderprotection.

In particular, there is a need to address remaining unmet medical needfor coverage of pneumococcal disease due to serotypes not found inPREVNAR 13® and potential for emergence of non PREVNAR 13® serotypes.The specific serotypes causing disease beyond the 13 in PREVNAR 13® varyby region, population, and may change over time due to acquisition ofantibiotic resistance, pneumococcal vaccine introduction and seculartrends of unknown origin. There is a need for immunogenic compositionsthat can be used to induce an immune response against additionalStreptococcus pneumoniae serotypes in humans and in particular inchildren less than 2 years old.

An object of the new immunogenic compositions of the present inventionis to provide for appropriate protection against S. pneumoniae serotypesnot found in PREVNAR 13®. In one aspect, an object of the immunogeniccompositions of the present invention is to provide for appropriateprotection against S. pneumoniae serotypes not found in PREVNAR®(heptavalent vaccine), SYNFLORIX® and/or PREVNAR 13® while maintainingan immune response against serotypes currently covered by said vaccines.

The phenomenon of antigenic competition (or interference) complicatesthe development of multi-valent vaccines. Antigenic interference refersto the observation that administering multiple antigens can result in adiminished response to certain antigens relative to the immune responseobserved when such antigens are administered individually. Itsoccurrence when making new combinations of antigens is unpredictable.

An object of the immunogenic compositions, kits and schedules ofadministration of the present invention is to provide for appropriateprotection against S. pneumoniae serotypes not found in PREVNAR 13®while maintaining an immune response against serotypes currently coveredby said vaccine and minimizing the risk of immune interference.

SUMMARY OF THE INVENTION

To meet these and other needs, the present invention relates to novelimmunogenic compositions, kits comprising the same and uses thereof. Thefollowing clauses describe some aspects and embodiments of theinvention.

One aspect of the invention relates to an immunogenic compositioncomprising at least one glycoconjugate selected from the groupconsisting of a glycoconjugate from S. pneumoniae serotype 15B, aglycoconjugate from S. pneumoniae serotype 22F, a glycoconjugate from S.pneumoniae serotype 33F, a glycoconjugate from S. pneumoniae serotype12F, a glycoconjugate from S. pneumoniae serotype 10A, a glycoconjugatefrom S. pneumoniae serotype 11A and a glycoconjugate from S. pneumoniaeserotype 8, wherein said composition is a 1, 2, 3, 4, 5, 6 or 7-valentpneumococcal conjugate composition.

In an aspect the invention provides a kit comprising: (a) a firstimmunogenic composition comprising said immunogenic composition; and (b)a second immunogenic composition comprising at least one glycoconjugatefrom a Streptococcus pneumoniae serotype selected from the groupconsisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F,23F, 22F and 33F.

In another aspect, the invention provides said immunogenic compositionfor simultaneous, concurrent, concomitant or sequential administration.

One aspect of the invention provides for said immunogenic compositionfor use in a vaccination schedule. In one embodiment, the vaccinationschedule is a single dose schedule. In another embodiment, thevaccination schedule is a multiple dose schedule.

In an aspect, said kit is for simultaneous, concurrent, concomitant orsequential administration of the first and second immunogeniccompositions.

Another aspect of the invention provides for said immunogeniccomposition or said kit for use as a medicament.

In one aspect of the invention, said immunogenic composition or said kitis for use as a vaccine.

In another aspect of the invention provides for said immunogeniccomposition or said kit for use in a method for preventing, treating orameliorating a bacterial infection, disease or condition in a subject.

Another aspect of the invention, said immunogenic composition or saidkit is for use in a method for preventing a bacterial infection, diseaseor condition in a subject.

One aspect of the invention provides for said immunogenic composition orsaid kit for use in a method to protect or treat a human susceptible topneumococcal infection, by means of administering said immunogeniccompositions via a systemic or mucosal route.

FIGURES

FIG. 1 shows a repeating polysaccharide structure of S. pneumoniaeserotype 8 (Pn-8) capsular polysaccharide.

FIG. 2 shows a repeating polysaccharide structure of S. pneumoniaeserotype 10A (Pn-10A) capsular polysaccharide.

FIG. 3 shows a repeating polysaccharide structure of S. pneumoniaeserotype 11A (Pn-11A) capsular polysaccharide.

FIG. 4 shows a repeating polysaccharide structure of S. pneumoniaeserotype 12F (Pn-12F) capsular polysaccharide.

FIG. 5 shows a repeating polysaccharide structure of S. pneumoniaeserotype 15B (Pn-15B) capsular polysaccharide.

FIG. 6 shows a repeating polysaccharide structure of S. pneumoniaeserotype 22F (Pn-22F) capsular polysaccharide.

FIG. 7 shows a repeating polysaccharide structure of S. pneumoniaeserotype 33F (Pn-33F) capsular polysaccharide.

FIG. 8 shows a representative process flow diagram for the activation(A) and conjugation (B) processes which can be used in the preparationof Pn-33F glycoconjugate.

FIG. 9 shows the effect on DO by varying amount of NCS in the TEMPO/NCSoxidation reaction.

FIG. 10 shows evaluation of Pn-12F glycoconjugates stability.

FIG. 11 Cross-Functional OPA Responses. A subset of 59 sera from adultsvaccinated with a 13 valent Pneumococcal Conjugate Vaccine (US Study6115A1-004; ClinicalTrials.gov Identifier: NCT00427895) was assessed inOPAs for the presence of functional antibodies against serotypes 9V, 9A,9L, and 9N. The percent of samples with OPA positive titer (i.e., ≥1:8)is indicated above each group. Geometric mean titers (GMT) are listed inthe x axis below each group.

FIG. 12 Cross-Functional OPA Responses of Sixty-six Matched pre/postSera. A subset of 66 matched pre- and post-vaccinated serum panel fromadults vaccinated with a 13 valent Pneumococcal Conjugate Vaccine (study6115A1-3005; ClinicalTrials.gov Identifier: NCT00546572) were assessedin OPAs for the presence of functional antibodies against serotypes 9V,9A, 9L, and 9N. The percent of samples with OPA positive titer (i.e.,≥1:8) is indicated above each group. Geometric mean titers (GMT) arelisted in the x axis below each group.

FIG. 13 Reverse cumulative distribution curves (RCDC) of pre and postImmunization—pneumococcal serotype 9V (Pn9V).

Reverse cumulative distribution curves of OPA titers to serotype 9V froma matched pre- and post-vaccination serum panel (N=66) vaccinated with a13 valent Pneumococcal Conjugate Vaccine (study 6115A1-3005;ClinicalTrials.gov Identifier: NCT00546572). The plots represent thepercent of sera with OPA positive titer (i.e., ≥1:8).

FIG. 14 Reverse cumulative distribution curves (RCDC) of pre and postImmunization—pneumococcal serotype 9A (Pn9A).

Reverse cumulative distribution curves of OPA titers to serotype 9A froma matched pre- and post-vaccination serum panel (N=66) vaccinated with a13 valent Pneumococcal Conjugate Vaccine (study 6115A1-3005;ClinicalTrials.gov Identifier: NCT00546572). The plots represent thepercent of sera with OPA positive titer (i.e., ≥1:8).

FIG. 15 Reverse cumulative distribution curves (RCDC) of pre and postImmunization—pneumococcal serotype 9L (Pn9L).

Reverse cumulative distribution curves of OPA titers to serotype 9L froma matched pre- and post-vaccination serum panel (N=66) vaccinated withwith a 13 valent Pneumococcal Conjugate Vaccine (study 6115A1-3005;ClinicalTrials.gov Identifier: NCT00546572). The plots represent thepercent of sera with OPA positive titer (i.e., ≥1:8).

FIG. 16 Reverse cumulative distribution curves (RCDC) of pre and postImmunization—pneumococcal serotype 9N (Pn9N).

Reverse cumulative distribution curves of OPA titers to serotype 9N froma matched pre- and post-vaccination serum panel (N=66) vaccinated withwith a 13 valent Pneumococcal Conjugate Vaccine (study 6115A1-3005;ClinicalTrials.gov Identifier: NCT00546572). The plots represent thepercent of sera with OPA positive titer (i.e., ≥1:8).

1. GLYCOCONJUGATES OF THE INVENTION

Immunogenic compositions of the present invention will typicallycomprise conjugated capsular saccharide antigens (also namedglycoconjugates), wherein the saccharides are derived from serotypes ofS. pneumoniae.

If the protein carrier is the same for 2 or more saccharides in thecomposition, the saccharides could be conjugated to the same molecule ofthe protein carrier (carrier molecules having 2 or more differentsaccharides conjugated to it) [see for instance WO2004/083251].

In a preferred embodiment though, the saccharides are each individuallyconjugated to different molecules of the protein carrier (each moleculeof protein carrier only having one type of saccharide conjugated to it).In said embodiment, the capsular saccharides are said to be individuallyconjugated to the carrier protein.

For the purposes of the invention the term ‘glycoconjugate’ indicates acapsular saccharide linked covalently to a carrier protein. In oneembodiment a capsular saccharide is linked directly to a carrierprotein. In a second embodiment a bacterial saccharide is linked to aprotein through a spacer/linker.

1.1 Carrier Protein of the Invention

A component of the glycoconjugate of the invention is a carrier proteinto which the saccharide is conjugated. The terms “protein carrier” or“carrier protein” or “carrier” may be used interchangeably herein.Carrier proteins should be amenable to standard conjugation procedures.

In a preferred embodiment, the carrier protein of the glycoconjugates isselected in the group consisting of: DT (Diphtheria toxin), TT (tetanustoxid) or fragment C of TT, CRM₁₉₇ (a nontoxic but antigenicallyidentical variant of diphtheria toxin), other DT mutants (such asCRM176, CRM228, CRM45 (Uchida et al. (1973) J. Biol. Chem.218:3838-3844), CRM9, CRM102, CRM103 or CRM107; and other mutationsdescribed by Nicholls and Youle in Genetically Engineered Toxins, Ed:Frankel, Maecel Dekker Inc. (1992); deletion or mutation of Glu-148 toAsp, Gin or Ser and/or Ala 158 to Gly and other mutations disclosed inU.S. Pat. Nos. 4,709,017 and 4,950,740; mutation of at least one or moreresidues Lys 516, Lys 526, Phe 530 and/or Lys 534 and other mutationsdisclosed in U.S. Pat. Nos. 5,917,017 and 6,455,673; or fragmentdisclosed in U.S. Pat. No. 5,843,711, pneumococcal pneumolysin (ply)(Kuo et al. (1995) Infect Immun 63:2706-2713) including ply detoxifiedin some fashion, for example dPLY-GMBS (WO 2004/081515, WO 2006/032499)or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE (sequences ofPhtA, PhtB, PhtD or PhtE are disclosed in WO 00/37105 and WO 00/39299)and fusions of Pht proteins, for example PhtDE fusions, PhtBE fusions,Pht A-E (WO 01/98334, WO 03/054007, WO 2009/000826), OMPC (meningococcalouter membrane protein), which is usually extracted from Neisseriameningitidis serogroup B (EP0372501), PorB (from N. meningitidis), PD(Haemophilus influenzae protein D; see, e.g., EP0594610 B), orimmunologically functional equivalents thereof, synthetic peptides(EP0378881, EP0427347), heat shock proteins (WO 93/17712, WO 94/03208),pertussis proteins (WO 98/58668, EP0471177), cytokines, lymphokines,growth factors or hormones (WO 91/01146), artificial proteins comprisingmultiple human CD4+ T cell epitopes from various pathogen derivedantigens (Falugi et al. (2001) Eur J Immunol 31:3816-3824) such as N19protein (Baraldoi et al. (2004) Infect Immun 72:4884-4887) pneumococcalsurface protein PspA (WO 02/091998), iron uptake proteins (WO 01/72337),toxin A or B of Clostridium difficile (WO 00/61761), transferrin bindingproteins, pneumococcal adhesion protein (PsaA), recombinant Pseudomonasaeruginosa exotoxin A (in particular non-toxic mutants thereof (such asexotoxin A bearing a substitution at glutamic acid 553 (Douglas et al.(1987) J. Bacteriol. 169(11):4967-4971)). Other proteins, such asovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA)or purified protein derivative of tuberculin (PPD) also can be used ascarrier proteins. Other suitable carrier proteins include inactivatedbacterial toxins such as cholera toxoid (e.g., as described in WO2004/083251), Escherichia coli LT, E. coli ST, and exotoxin A from P.aeruginosa.

In a preferred embodiment, the carrier protein of the glycoconjugates isindependently selected from the group consisting of TT, DT, DT mutants(such as CRM₁₉₇), H. influenzae protein D, PhtX, PhtD, PhtDE fusions(particularly those described in WO 01/98334 and WO 03/054007),detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, toxin A or B ofC. difficile and PsaA.

In an embodiment, the carrier protein of the glycoconjugates of theinvention is DT (Diphtheria toxoid). In another embodiment, the carrierprotein of the glycoconjugates of the invention is TT (tetanus toxid).

In another embodiment, the carrier protein of the glycoconjugates of theinvention is PD (H. influenzae protein D; see, e.g., EP0594610 B).

In a preferred embodiment, the capsular saccharides of the invention areconjugated to CRM₁₉₇ protein. The CRM₁₉₇ protein is a nontoxic form ofdiphtheria toxin but is immunologically indistinguishable from thediphtheria toxin. CRM₁₉₇ is produced by Corynebacterium diphtheriaeinfected by the nontoxigenic phage β197^(tox−) created bynitrosoguanidine mutagenesis of the toxigenic corynephage beta (Uchidaet al. (1971) Nature New Biology 233:8-11). The CRM₁₉₇ protein has thesame molecular weight as the diphtheria toxin but differs therefrom by asingle base change (guanine to adenine) in the structural gene. Thissingle base change causes an amino acid substitution (glutamic acid forglycine) in the mature protein and eliminates the toxic properties ofdiphtheria toxin. The CRM₁₉₇ protein is a safe and effective T-celldependent carrier for saccharides. Further details about CRM₁₉₇ andproduction thereof can be found, e.g., in U.S. Pat. No. 5,614,382.

In an embodiment, the capsular saccharides of the invention areconjugated to CRM₁₉₇ protein or the A chain of CRM₁₉₇ (see CN103495161).In an embodiment, the capsular saccharides of the invention areconjugated the A chain of CRM₁₉₇ obtained via expression by geneticallyrecombinant E. coli (see CN103495161). In an embodiment, the capsularsaccharides of the invention are all conjugated to CRM₁₉₇. In anembodiment, the capsular saccharides of the invention are all conjugatedto the A chain of CRM₁₉₇.

Accordingly, in frequent embodiments, the glycoconjugates of theinvention comprise CRM₁₉₇ as the carrier protein, wherein the capsularpolysaccharide is covalently linked to CRM₁₉₇.

1.2 Capsular Saccharide of the Invention

The term “saccharide” throughout this specification may indicatepolysaccharide or oligosaccharide and includes both. In frequentembodiments, the saccharide is a polysaccharide, in particular a S.pneumoniae capsular polysaccharide.

Capsular polysaccharides are prepared by standard techniques known tothose of ordinary skill in the art.

In the present invention, capsular polysaccharides may be prepared,e.g., from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14,15B, 18C, 19A, 19F, 22F, 23F and 33F of S. pneumoniae. Typicallycapsular polysaccharides are produced by growing each S. pneumoniaeserotype in a medium (e.g. in a soy-based medium), the polysaccharidesare then prepared from the bacteria culture. Bacterial strains of S.pneumoniae used to make the respective polysaccharides that are used inthe glycoconjugates of the invention may be obtained from establishedculture collections or clinical specimens.

The population of the organism (each S. pneumoniae serotype) is oftenscaled up from a seed vial to seed bottles and passaged through one ormore seed fermentors of increasing volume until production scalefermentation volumes are reached. At the end of the growth cycle thecells are lysed and the lysate broth is then harvested for downstream(purification) processing (see for example WO 2006/110381, WO2008/118752, and U.S. Patent App. Pub. Nos. 2006/0228380, 2006/0228381,2008/0102498 and 2008/0286838).

The individual polysaccharides are typically purified throughcentrifugation, precipitation, ultra-filtration, and/or columnchromatography (see for example WO 2006/110352 and WO 2008/118752).

Purified polysaccharides may be activated (e.g., chemically activated)to make them capable of reacting (e.g., with the eTEC spacer) and thenincorporated into glycoconjugates of the invention, as further describedherein.

S. pneumoniae capsular polysaccharides comprise repeatingoligosaccharide units which may contain up to 8 sugar residues.

In an embodiment, capsular saccharide of the invention may be oneoligosaccharide unit or a shorter than native length saccharide chain ofrepeating oligosaccharide units. In an embodiment, capsular saccharideof the invention is one repeating oligosaccharide unit of the relevantserotype.

In an embodiment, capsular saccharide of the invention may beoligosaccharides. Oligosaccharides have a low number of repeat units(typically 5-15 repeat units) and are typically derived synthetically orby hydrolysis of polysaccharides.

Preferably though, all of the capsular saccharides of the presentinvention and in the immunogenic compositions of the present inventionare polysaccharides. High molecular weight capsular polysaccharides areable to induce certain antibody immune responses due to the epitopespresent on the antigenic surface. The isolation and purification of highmolecular weight capsular polysaccharides is preferably contemplated foruse in the conjugates, compositions and methods of the presentinvention.

In some embodiments, the purified polysaccharides before conjugationhave a molecular weight of between 10 kDa and 4,000 kDa. In other suchembodiments, the polysaccharide has a molecular weight of between 50 kDaand 4,000 kDa. In further such embodiments, the polysaccharide has amolecular weight of between 50 kDa and 3,500 kDa; between 50 kDa and3,000 kDa; between 50 kDa and 2,500 kDa; between 50 kDa and 2,000 kDa;between 50 kDa and 1,750 kDa; between 50 kDa and 1,500 kDa; between 50kDa and 1,250 kDa; between 50 kDa and 1,000 kDa; between 50 kDa and 750kDa; between 50 kDa and 500 kDa; between 100 kDa and 4,000 kDa; between100 kDa and 3,500 kDa; 100 kDa and 3,000 kDa; 100 kDa and 2,500 kDa; 100kDa and 2,250 kDa; between 100 kDa and 2,000 kDa; between 100 kDa and1,750 kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa;between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100kDa and 500 kDa; between 200 kDa and 4,000 kDa; between 200 kDa and3,500 kDa; between 200 kDa and 3,000 kDa; between 200 kDa and 2,500 kDa;between 200 kDa and 2,250 kDa; between 200 kDa and 2,000 kDa; between200 kDa and 1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDaand 1,250 kDa; between 200 kDa and 1,000 kDa; between 200 kDa and 750kDa; or between 200 kDa and 500 kDa. Any whole number integer within anyof the above ranges is contemplated as an embodiment of the disclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described herein,polysaccharide can be subjected to sizing techniques before conjugation.Mechanical or chemical sizing maybe employed. Chemical hydrolysis maybeconducted using acetic acid. Mechanical sizing maybe conducted usingHigh Pressure Homogenization Shearing. The molecular weight rangesmentioned above refer to purified polysaccharides before conjugation(e.g., before activation).

In a preferred embodiment the purified polysaccharides, are capsularpolysaccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A,12F, 14, 15B, 18C, 19A, 19F, 22F, 23F or 33F of S. pneumoniae, whereinthe capsular polysaccharide has a molecular weight falling within one ofthe molecular weight ranges as described here above.

As used herein, the term “molecular weight” of polysaccharide or ofcarrier protein-polysaccharide conjugate refers to molecular weightcalculated by size exclusion chromatography (SEC) combined withmultiangle laser light scattering detector (MALLS).

In some embodiments, the pneumococcal saccharides from serotypes 9V,18C, 11A, 15B, 22F and/or 33F of the invention are O-acetylated. In someembodiments, the pneumococcal saccharides from serotypes 9V, 11A, 15B,22F and/or 33F of the invention are O-acetylated.

The purified polysaccharides described herein are chemically activatedto make the saccharides capable of reacting with the carrier protein.These pneumococcal conjugates are prepared by separate processes andformulated into a single dosage formulation as described below.

1.2.1 Pneumococcal Polysaccharide from S. pneumoniae Serotypes 1, 3, 4,5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F

Capsular saccharides from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B,7F, 9V, 14, 18C, 19A, 19F and 23F may be prepared by standard techniquesknown to those of ordinary skill in the art (see for example WO2006/110381). Capsular polysaccharides can be produced by growing eachS. pneumoniae serotype in a medium; at the end of the growth cycle thecells are lysed and the lysate broth is then harvested for downstream(purification) processing. The individual polysaccharides are typicallypurified through centrifugation, precipitation, ultra-filtration, and/orcolumn chromatography (see for example WO 2006/110352 and WO2008/118752). Purified polysaccharides may be further processed asfurther described herein to prepare glycoconjugates of the invention.

In some embodiments, the purified polysaccharides from S. pneumoniaeserotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and/or 23Fbefore conjugation have a molecular weight of between 10 kDa and 4,000kDa. In other such embodiments, the polysaccharide has a molecularweight of between 50 kDa and 4,000 kDa; between 50 kDa and 3,000 kDa orbetween 50 kDa and 2,000 kDa. In further such embodiments, thepolysaccharide has a molecular weight of between between 50 kDa and3,500 kDa; between 50 kDa and 3,000 kDa; between 50 kDa and 2,500 kDa;between 50 kDa and 2,000 kDa; 50 kDa and 1,750 kDa; between 50 kDa and1,500 kDa; between 50 kDa and 1,250 kDa; between 50 kDa and 1,000 kDa;between 50 kDa and 750 kDa; between 50 kDa and 500 kDa; between 100 kDaand 4,000 kDa; between 100 kDa and 3,500 kDa; between 100 kDa and 3,000kDa; between 100 kDa and 2,500 kDa; between 100 kDa and 2,000 kDa;between 100 kDa and 1,750 kDa; between 100 kDa and 1,500 kDa; between100 kDa and 1,250 kDa; between 100 kDa and 1,000 kDa; between 100 kDaand 750 kDa; between 100 kDa and 500 kDa; between 200 kDa and 4,000 kDa;between 200 kDa and 3,500 kDa; between 200 kDa and 3,000 kDa; between200 kDa and 2,500 kDa; between 200 kDa and 2,000 kDa; between 200 kDaand 1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDa and 1,250kDa; between 200 kDa and 1,000 kDa; between 200 kDa and 750 kDa; orbetween 200 kDa and 500 kDa. Any whole number integer within any of theabove ranges is contemplated as an embodiment of the disclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described herein,polysaccharide can be subjected to sizing techniques before conjugation.The molecular weight ranges mentioned above refer to purifiedpolysaccharides before conjugation (e.g., before activation) after aneventual sizing step.

In some embodiments, the pneumococcal saccharides from serotypes 9Vand/or 18C of the invention are O-acetylated. In some embodiments, thepneumococcal saccharide from serotype 9V of the invention isO-acetylated and the pneumococcal saccharide from serotype 18C of theinvention is de-O-acetylated.

1.2.2 Pneumococcal Polysaccharide Serotype 8

The polysaccharide repeating unit of serotype 8 consists of a lineartetrasaccharide unit with one glucuronic acid (GlcpA), twoglucopyranoses (Glcp) and one galactopyranose (Galp) (Jones et al.(1957) The Journal of the American Chemical Society. 79(11):2787-2793).All four monosaccharides are linked via 1,4-linkages as shown at FIG. 1.

Serotype 8 saccharides can be obtained directly from bacteria usingisolation procedures known to one of ordinary skill in the art (see forexample methods disclosed in U.S. Patent App. Pub. Nos. 2006/0228380,2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340, and 2008/0102498and WO 2008/118752). In addition, they can be produced using syntheticprotocols.

Serotype 8 S. pneumoniae strains may be obtained from establishedculture collections (such as for example the Streptococcal ReferenceLaboratory (Centers for Disease Control and Prevention, Atlanta, Ga.))or clinical specimens.

In some embodiments, the purified polysaccharides from S. pneumoniaeserotype 8 before conjugation have a molecular weight of between 10 kDaand 2,000 kDa. In one embodiment, the capsular polysaccharide has amolecular weight of between 50 kDa and 1,000 kDa. In another embodiment,the capsular polysaccharide has a molecular weight of between 70 kDa and900 kDa. In another embodiment, the capsular polysaccharide has amolecular weight of between 100 kDa and 800 kDa.

In further embodiments, the capsular polysaccharide has a molecularweight of 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa;150 kDa to 600 kDa; 150 kDa to 500 kDa; 150 kDa to 400 kDa; 200 kDa to600 kDa; 200 kDa to 500 kDa; 200 kDa to 400 kDa; 250 kDa to 600; 250 kDato 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa; 300 kDa to 600 kDa;300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDa to 600 kDa; 500 kDa to600 kDa; and similar desired molecular weight ranges. Any whole numberinteger within any of the above ranges is contemplated as an embodimentof the disclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described herein,polysaccharide can be subjected to sizing techniques before conjugation.The molecular weight ranges mentioned above refer to purifiedpolysaccharides before conjugation (e.g., before activation) after aneventual sizing step.

1.2.3 Pneumococcal Polysaccharide Serotype 10A

The polysaccharide repeating unit of serotype 10A consists of a branchedhexasaccharide repeat unit with two galactofuranoses (Gal_(f)), threegalactopyranoses (Gal_(p)), one N-acetylgalactosamine (Gal_(p)NAc) and abackbone phosphoribitol (Jones, C. (2005) Carbohydrate Research269(1):175-181). There are two branching monosaccharides at theβ-GalpNAc moiety (a β-3-Galp and a β-6-Galf) as shown at FIG. 2.

Serotype 10A saccharides can be obtained directly from bacteria usingisolation procedures known to one of ordinary skill in the art (see forexample methods disclosed in U.S. Patent App. Pub. Nos. 2006/0228380,2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340, and 2008/0102498and WO 2008/118752). In addition, they can be produced using syntheticprotocols.

Serotype 10A S. pneumoniae strains may be obtained from establishedculture collections (such as for example the Streptococcal ReferenceLaboratory (Centers for Disease Control and Prevention, Atlanta, Ga.))or clinical specimens.

In some embodiments, the purified polysaccharides from S. pneumoniaeserotype 10A before conjugation have a molecular weight of between 10kDa and 2,000 kDa. In one embodiment, the capsular polysaccharide has amolecular weight of between 50 kDa and 1,000 kDa. In another embodiment,the capsular polysaccharide has a molecular weight of between 70 kDa and900 kDa. In another embodiment, the capsular polysaccharide has amolecular weight of between 100 kDa and 800 kDa.

In further embodiments, the capsular polysaccharide has a molecularweight of 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa;150 kDa to 600 kDa; 150 kDa to 500 kDa; 150 kDa to 400 kDa; 200 kDa to600 kDa; 200 kDa to 500 kDa; 200 kDa to 400 kDa; 250 kDa to 600 kDa; 250kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa; 300 kDa to 600kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDa to 600 kDa; 500 kDato 600 kDa; and similar desired molecular weight ranges. Any wholenumber integer within any of the above ranges is contemplated as anembodiment of the disclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described herein,polysaccharide can be subjected to sizing techniques before conjugation.The molecular weight ranges mentioned above refer to purifiedpolysaccharides before conjugation (e.g., before activation) after aneventual sizing step.

1.2.4 Pneumococcal Polysaccharide Serotype 11A

The polysaccharide repeating unit of serotype 11A consists of a lineartetrasaccharide backbone (two galactopyranoses (Gal_(p)) and twoglucopyranose (Glc_(p))) and a pendent phosphoglycerol (Richards et al.(1988) Adv. Exp. Med. Biol. 228:595-597), as shown at FIG. 3. Thepolysaccharide is O-acetylated at multiple locations and, based on thereported data in the literature (Calix et al. (2011) J Bacteriol.193(19):5271-5278), the total amount of O-acetylation in 11Apolysaccharide is about 2.6 O-acetyl groups per polysaccharide repeatunit.

Serotype 11A saccharides can be obtained directly from bacteria usingisolation procedures known to one of ordinary skill in the art (see forexample methods disclosed in U.S. Patent App. Pub. Nos. 2006/0228380,2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340, and 2008/0102498and WO 2008/118752). In addition, they can be produced using syntheticprotocols.

Serotype 11A S. pneumoniae strains may be obtained from establishedculture collections (such as for example the Streptococcal ReferenceLaboratory (Centers for Disease Control and Prevention, Atlanta, Ga.))or clinical specimens.

The isolated serotype 11A capsular polysaccharide obtained bypurification of serotype 11A polysaccharide from the S. pneumoniaelysate and optionally sizing of the purified polysaccharide may becharacterized by different attributes including, for example, themolecular weight (MW) and the mM of acetate per mM of said serotype 11Acapsular polysaccharide.

In some embodiments, the purified polysaccharides from S. pneumoniaeserotype 11A before conjugation have a molecular weight of between 10kDa and 2,000 kDa. In one embodiment, the capsular polysaccharide has amolecular weight of between 50 kDa and 1,000 kDa. In another embodiment,the capsular polysaccharide has a molecular weight of between 70 kDa and900 kDa. In another embodiment, the capsular polysaccharide has amolecular weight of between 100 kDa and 800 kDa.

In further embodiments, the capsular polysaccharide has a molecularweight of 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa;100 kDa to 300 kDa; 100 kDa to 200 kDa; 150 kDa to 600 kDa; 150 kDa to500 kDa; 150 kDa to 400 kDa; 150 kDa to 300 kDa; 150 kDa to 200 kDa; 200kDa to 600 kDa; 200 kDa to 500 kDa; 200 kDa to 400 kDa; 250 kDa to 600kDa; 250 kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa; 300 kDato 600 kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDa to 600 kDa;500 kDa to 600 kDa; and similar desired molecular weight ranges. Anywhole number integer within any of the above ranges is contemplated asan embodiment of the disclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described herein,polysaccharide can be subjected to sizing techniques before conjugation.The molecular weight ranges mentioned above refer to purifiedpolysaccharides before conjugation (e.g., before activation) after aneventual sizing step.

In an embodiment, the size of the purified serotype 11A polysaccharideis reduced by high pressure homogenization. High pressure homogenizationachieves high shear rates by pumping the process stream through a flowpath with sufficiently small dimensions. The shear rate is increased byusing a larger applied homogenization pressure, and exposure time can beincreased by recirculating the feed stream through the homogenizer.

The high pressure homogenization process is particularly appropriate forreducing the size of the purified serotype 11A polysaccharide whilepreserving the structural features of the polysaccharide, such as thepresence of O-acetyl groups.

The presence of O-acetyl in a purified, isolated or activated serotype11A capsular polysaccharide or in a serotype 11A polysaccharide-carrierprotein conjugate is expressed as the number of mM of acetate per mM ofsaid polysaccharide or as the number of O-acetyl group perpolysaccharide repeating unit.

In a preferred embodiment, the purified polysaccharides from S.pneumoniae serotype 11A has at least 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4or 1.6 μmol acetate per μmol of said serotype 11A capsularpolysaccharide.

1.2.5 Pneumococcal Polysaccharide Serotype 12F

The polysaccharide repeating unit of serotype 12F consists of a lineartrisaccharide backbone (one N-acetylfucosamine (Fuc_(p)NAc), oneN-acetylgalactosamine (Gal_(p)NAc) and one N-acetylmannuronic acid(Man_(p)NAcA)) with two branches: a pendant α-galactopyranose (Gal_(p))linked at C3 of Fuc_(p)NAc and an α-Glc_(p)-(1-2)-α-Glc_(p) disaccharidebranch linked at C3 of Man_(p)NAcA (Leontein et al. (1983) CarbohydrateResearch 114(2):257-266.) as shown at FIG. 4.

Serotype 12F Streptococcus pneumoniae strains may be obtained fromestablished culture collections (such as for example the StreptococcalReference Laboratory (Centers for Disease Control and Prevention,Atlanta, Ga.)) or clinical specimens.

Capsular saccharides from S. pneumoniae serotype 12F are prepared bystandard techniques known to those of ordinary skill in the art.Typically capsular polysaccharides are produced by growing each S.pneumoniae serotype in a medium (e.g., in a soy-based medium), thepolysaccharides are then prepared from the bacteria culture. Thepopulation of the organism (S. pneumoniae serotype 12F) is often scaledup from a seed vial to seed bottles and passaged through one or moreseed fermentors of increasing volume until production scale fermentationvolumes are reached. At the end of the growth cycle the cells are lysedand the lysate broth is then harvested for downstream (purification)processing (see for example WO 2006/110381 and WO 2008/118752, U.S.Patent App. Pub. Nos. 2006/0228380, 2006/0228381, 2008/0102498 andUS2008/0286838). The polysaccharides are typically purified throughcentrifugation, precipitation, ultra-filtration, and/or columnchromatography (see for example WO 2006/110352 and WO 2008/118752).

Purified polysaccharides from serotype 12F may be activated (e.g.,chemically activated) to make them capable of reacting and thenincorporated into glycoconjugates of the invention, as further describedherein.

In some embodiments, the purified polysaccharides from S. pneumoniaeserotype 12F before conjugation have a molecular weight of between 10kDa and 2,000 kDa. In one embodiment, the capsular polysaccharide has amolecular weight of between 50 kDa and 1,000 kDa. In another embodiment,the capsular polysaccharide has a molecular weight of between 50 kDa and300 kDa. In another embodiment, the capsular polysaccharide has amolecular weight of between 70 kDa and 300 kDa. In further embodiments,the capsular polysaccharide has a molecular weight of 90 kDa to 250 kDa;90 kDa to 150 kDa; 90 kDa to 120 kDa; 80 kDa to 120 kDa; 70 kDa to 100kDa; 70 kDa to 110 kDa; 70 kDa to 120 kDa; 70 kDa to 130 kDa; 70 kDa to140 kDa; 70 kDa to 150 kDa; 70 kDa to 160 kDa; 80 kDa to 110 kDa; 80 kDato 120 kDa; 80 kDa to 130 kDa; 80 kDa to 140 kDa; 80 kDa to 150 kDa; 80kDa to 160 kDa; 90 kDa to 110 kDa; 90 kDa to 120 kDa; 90 kDa to 130 kDa;90 kDa to 140 kDa; 90 kDa to 150 kDa; 90 kDa to 160 kDa; 100 kDa to 120kDa; 100 kDa to 130 kDa; 100 kDa to 140 kDa; 100 kDa to 150 kDa; 100 kDato 160 kDa; and similar desired molecular weight ranges. Any wholenumber integer within any of the above ranges is contemplated as anembodiment of the disclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described herein,polysaccharide can be subjected to sizing techniques before conjugation.The molecular weight ranges mentioned above refer to purifiedpolysaccharides before conjugation (e.g., before activation) after aneventual sizing step.

1.2.6 Pneumococcal Polysaccharide Serotype 15B

As shown at FIG. 5, the polysaccharide repeating unit of serotype 15Bconsists of a branched trisaccharide backbone (one N-acetylglucosamine(Glc_(p)NAc), one galactopyranose (Gal_(p)) and one glucopyranose(Glc_(p))) with an αGal_(p)-βGal_(p) disaccharide branch linked to theC4 hydroxyl group of Glc_(p)NAc. The phosphoglycerol is linked to the C3hydroxyl group of the βGal_(p) residue in the disaccharide branch (Joneset al. (2005) Carbohydrate Research 340(3):403-409). Capsularpolysaccharide from serotype 15C serotype has the identical backbonestructure as serotype 15B but lacks the O-acetylation.

Serotype 15B polysaccharides can be obtained directly from bacteriausing isolation procedures known to one of ordinary skill in the art(see for example methods disclosed in U.S. Patent App. Pub. Nos.2006/0228380, 2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340,and 2008/0102498 and WO 2008/118752). They can also be produced usingsynthetic protocols known to the man skilled in the art.

Serotype 15B S. pneumoniae strains may be obtained from establishedculture collections (such as for example the American Type CultureCollection (ATCC, Manassas, Va. USA) (e.g., deposit strain No.ATCC10354) or the Streptococcal Reference Laboratory (Centers forDisease Control and Prevention, Atlanta, Ga. USA)) or from clinicalspecimens.

The bacterial cells are grown in a medium, preferably in a soy basedmedium. Following fermentation of bacterial cells that produce S.pneumoniae serotype 15B capsular polysaccharides, the bacterial cellsare lysed to produce a cell lysate. The serotype 15B polysaccharide maythen be isolated from the cell lysate using purification techniquesknown in the art, including the use of centrifugation, depth filtration,precipitation, ultra-filtration, treatment with activate carbon,diafiltration and/or column chromatography (see, for example, U.S.Patent App. Pub. Nos. 2006/0228380, 2006/0228381, 2007/0184071,2007/0184072, 2007/0231340, and 2008/0102498 and WO 2008/118752). Thepurified serotype 15B capsular polysaccharide can then be used for thepreparation of immunogenic conjugates.

The isolated serotype 15B capsular polysaccharide obtained bypurification of serotype 15B polysaccharide from the S. pneumoniaelysate and optionally sizing of the purified polysaccharide can becharacterized by different parameters including, for example, themolecular weight (MW), the mM of acetate per mM of said serotype 15Bcapsular polysaccharide and the mM of glycerol per mM of said serotype15B capsular polysaccharide.

Preferably, in order to generate 15B conjugates with advantageousfilterability characteristics and/or yields, sizing of thepolysaccharide to a target molecular weight range is performed prior tothe conjugation to a carrier protein. Advantageously, the size of thepurified serotype 15B polysaccharide is reduced while preservingcritical features of the structure of the polysaccharide such as forexample the presence of O-acetyl groups. Preferably, the size of thepurified serotype 15B polysaccharide is reduced by mechanicalhomogenization.

In a preferred embodiment, the size of the purified serotype 15Bpolysaccharide is reduced by high pressure homogenization. High pressurehomogenization achieves high shear rates by pumping the process streamthrough a flow path with sufficiently small dimensions. The shear rateis increased by using a larger applied homogenization pressure, andexposure time can be increased by recirculating the feed stream throughthe homogenizer.

The high pressure homogenization process is particularly appropriate forreducing the size of the purified serotype 15B polysaccharide whilepreserving the structural features of the polysaccharide, such as thepresence of O-acetyl groups.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 5 kDa and 500 kDa, between50 kDa and 500 kDa, between 50 kDa and 450 kDa, between 100 kDa and 400kDa, and between 100 kDa and 350 kDa. In a preferred embodiment, theisolated serotype 15B capsular polysaccharide has a molecular weightbetween 100 kDa and 350 kDa. In a preferred embodiment, the isolatedserotype 15B capsular polysaccharide has a molecular weight between 100kDa and 300 kDa. In a preferred embodiment, the isolated serotype 15Bcapsular polysaccharide has a molecular weight between 150 kDa and 300kDa. In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 150 kDa and 350 kDa. Infurther embodiments, the capsular polysaccharide has a molecular weightof 100 kDa to 500 kDa; 100 kDa to 400 kDa; 100 kDa to 300 kDa; 100 kDato 200 kDa; 150 kDa to 500 kDa; 150 kDa to 400 kDa; 150 kDa to 300 kDa;150 kDa to 200 kDa; 200 kDa to 500 kDa; 200 kDa to 400 kDa; 250 kDa to500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa; 300 kDa to 500 kDa; 300kDa to 400 kDa; and similar desired molecular weight ranges. Any wholenumber integer within any of the above ranges is contemplated as anembodiment of the disclosure.

Serotype 15B polysaccharide is O-acetylated and the total amount ofO-acetylation is approximately 0.8-0.9 O-acetyl groups perpolysaccharide repeating unit. The degree of O-acetylation of thepolysaccharide can be determined by any method known in the art, forexample, by proton NMR (see for example Lemercinier et al. (1996)Carbohydrate Research 296:83-96; Jones et al. (2002) J. Pharmaceuticaland Biomedical Analysis 30:1233-1247; WO 2005/033148 and WO 00/56357).Another commonly used method is described in Hestrin, S. (1949) J. Biol.Chem. 180:249-261. Preferably, the presence of O-acetyl groups isdetermined by ion-HPLC analysis.

The presence of O-acetyl in a purified, isolated or activated serotype15B capsular polysaccharide or in a serotype 15B polysaccharide-carrierprotein conjugate is expressed as the number of mM of acetate per mM ofsaid polysaccharide or as the number of O-acetyl group perpolysaccharide repeating unit.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or0.8 mM acetate per mM of said serotype 15B capsular polysaccharide. In apreferred embodiment, the isolated serotype 15B capsular polysaccharidecomprises at least 0.5, 0.6 or 0.7 mM acetate per mM of said serotype15B capsular polysaccharide. In a preferred embodiment, the isolatedserotype 15B capsular polysaccharide comprises at least 0.6 mM acetateper mM of said serotype 15B capsular polysaccharide. In a preferredembodiment, the isolated serotype 15B capsular polysaccharide comprisesat least 0.7 mM acetate per mM of said serotype 15B capsularpolysaccharide.

The presence of glycerolphosphate side chains is determined bymeasurement of glycerol using high performance anion exchangechromatography with pulsed amperometric detection (HPAEC-PAD) after itsrelease by treatment of the polysaccharide with hydrofluoric acid (HF).The presence of glycerol in a purified, isolated or activated serotype15B polysaccharide or in a serotype 15B polysaccharide-carrier proteinconjugate is expressed as the number of mM of glycerol per mM ofserotype 15B polysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or0.8 mM glycerol per mM of said serotype 15B capsular polysaccharide. Ina preferred embodiment, the isolated serotype 15B capsularpolysaccharide comprises at least 0.5, 0.6 or 0.7 mM glycerol per mM ofsaid serotype 15B capsular polysaccharide. In a preferred embodiment,the isolated serotype 15B capsular polysaccharide comprises at least 0.6mM glycerol per mM of said serotype 15B capsular polysaccharide. In apreferred embodiment, the isolated serotype 15B capsular polysaccharidecomprises at least 0.7 mM glycerol per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 100 kDa and 350 kDa andcomprises at least 0.6 mM acetate per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 100 kDa and 350 kDa andcomprises at least 0.6 mM glycerol per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 150 kDa and 300 kDa andcomprises at least 0.6 mM acetate per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 150 kDa and 300 kDa andcomprises at least 0.6 mM glycerol per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 150 kDa and 350 kDa andcomprises at least 0.6 mM acetate per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 150 kDa and 350 kDa andcomprises at least 0.6 mM glycerol per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide comprises at least 0.6 mM acetate per mM of said serotype15B capsular polysaccharide and at least 0.6 mM glycerol per mM of saidserotype 15B capsular polysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 100 kDa and 350 kDa andcomprises at least 0.6 mM acetate per mM of said serotype 15B capsularpolysaccharide and at least 0.6 mM glycerol per mM of said serotype 15Bcapsular polysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 150 kDa and 300 kDa andcomprises at least 0.6 mM acetate per mM of said serotype 15B capsularpolysaccharide and at least 0.6 mM glycerol per mM of said serotype 15Bcapsular polysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 150 kDa and 350 kDa andcomprises at least 0.6 mM acetate per mM of said serotype 15B capsularpolysaccharide and at least 0.6 mM glycerol per mM of said serotype 15Bcapsular polysaccharide.

1.2.7 Pneumococcal Polysaccharide Serotype 22F

As shown at FIG. 6, the polysaccharide repeating unit of serotype 22Fconsists of a branched pentasaccharide backbone (one glucuronic acid(Glc_(p)A), one glucopyranose (Glc_(p)), one galactofuranose (Gal_(f))and two rhamnopyranoses (Rha_(p))) with a αGlc_(p) branch linked to theC3 hydroxyl group of βRha_(p) (Richards et al. (1989) Canadian Journalof Chemistry 67(6):1038-1050). Approximately 80% of the C2 hydroxylgroups of the βRha_(p) residue in the polysaccharide repeating unit areO-acetylated.

Serotype 22F polysaccharides can be obtained directly from bacteriausing isolation procedures known to one of ordinary skill in the art(see for example methods disclosed in U.S. Patent App. Pub. Nos.2006/0228380, 2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340,and 2008/0102498 and WO 2008/118752). In addition, they can be producedusing synthetic protocols.

Serotype 22F S. pneumoniae strains may be obtained from establishedculture collections (such as for example the Streptococcal ReferenceLaboratory (Centers for Disease Control and Prevention, Atlanta, Ga.))or clinical specimens.

The isolated serotype 22F capsular polysaccharide obtained bypurification of serotype 22F polysaccharide from the S. pneumoniaelysate and optionally sizing of the purified polysaccharide can becharacterized by different parameters including, for example, themolecular weight (MW) and the mM of acetate per mM of said serotype 22Fcapsular polysaccharide.

Preferably, in order to generate serotype 22F conjugates withadvantageous filterability characteristics and/or yields, sizing of thepolysaccharide to a target molecular weight range is performed prior tothe conjugation to a carrier protein. Advantageously, the size of thepurified serotype 22F polysaccharide is reduced while preservingcritical features of the structure of the polysaccharide such as forexample the presence of O-acetyl group. Preferably, the size of thepurified serotype 22F polysaccharide is reduced by mechanicalhomogenization.

In a preferred embodiment, the size of the purified polysaccharide isreduced by high pressure homogenization. High pressure homogenizationachieves high shear rates by pumping the process stream through a flowpath with sufficiently small dimensions. The shear rate is increased byusing a larger applied homogenization pressure, and exposure time can beincreased by recirculating the feed stream through the homogenizer.

The high pressure homogenization process is particularly appropriate forreducing the size of the purified serotype 22F polysaccharide whilepreserving the structural features of the polysaccharide, such as thepresence of O-acetyl groups.

In some embodiments, the purified polysaccharides from S. pneumoniaeserotype 22F before conjugation have a molecular weight of between 10kDa and 2,000 kDa. In one embodiment, the capsular polysaccharide has amolecular weight of between 50 kDa and 1,000 kDa. In another embodiment,the capsular polysaccharide has a molecular weight of between 70 kDa to900 kDa. In another embodiment, the capsular polysaccharide has amolecular weight of between 100 kDa to 800 kDa. In another embodiment,the capsular polysaccharide has a molecular weight of between 200 kDa to600 kDa. In another embodiment, the capsular polysaccharide has amolecular weight of between 400 kDa to 700 kDa.

In further embodiments, the capsular polysaccharide has a molecularweight of 100 kDa to 1,000 kDa; 100 kDa to 900 kDa; 100 kDa to 800 kDa;100 kDa to 700 kDa; 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to400 kDa; 100 kDa to 300 kDa; 150 kDa to 1,000 kDa; 150 kDa to 900 kDa;150 kDa to 800 kDa; 150 kDa to 700 kDa; 150 kDa to 600 kDa; 150 kDa to500 kDa; 150 kDa to 400 kDa; 150 kDa to 300 kDa; 200 kDa to 1,000 kDa;200 kDa to 900 kDa; 200 kDa to 800 kDa; 200 kDa to 700 kDa; 200 kDa to600 kDa; 200 kDa to 500 kDa; 200 kDa to 400 kDa; 200 kDa to 300 kDa; 250kDa to 1,000 kDa; 250 kDa to 900 kDa; 250 kDa to 800 kDa; 250 kDa to 700kDa; 250 kDa to 600 kDa; 250 kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDato 350 kDa; 300 kDa to 1,000 kDa; 300 kDa to 900 kDa; 300 kDa to 800kDa; 300 kDa to 700 kDa; 300 kDa to 600 kDa; 300 kDa to 500 kDa; 300 kDato 400 kDa; 400 kDa to 1,000 kDa; 400 kDa to 900 kDa; 400 kDa to 800kDa; 400 kDa to 700 kDa; 400 kDa to 600 kDa; 500 kDa to 600 kDa; andsimilar desired molecular weight ranges. Any whole number integer withinany of the above ranges is contemplated as an embodiment of thedisclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described hereabove, 22Fpolysaccharide can be subjected to sizing techniques before conjugation.The molecular weight ranges mentioned above refer to purifiedpolysaccharides before conjugation (e.g., before activation) after aneventual sizing step.

The degree of O-acetylation of the polysaccharide can be determined byany method known in the art, for example, by proton NMR (Lemercinier etal. (1996) Carbohydrate Research 296:83-96; Jones et al. (2002) J.Pharmaceutical and Biomedical Analysis 30:1233-1247; WO 2005/033148 andWO 00/56357). Another commonly used method is described in Hestrin, S.(1949) J. Biol. Chem. 180:249-261. Preferably, the presence of O-acetylgroups is determined by ion-HPLC analysis.

The presence of O-acetyl in a purified, isolated or activated serotype22F capsular polysaccharide or in a serotype 22F polysaccharide-carrierprotein conjugate is expressed as the number of mM of acetate per mM ofsaid polysaccharide or as the number of O-acetyl group perpolysaccharide repeating unit.

In a preferred embodiment, the purified polysaccharides from S.pneumoniae serotype 22F has at least 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4or 1.6, μmol acetate per μmol of said serotype 22F capsularpolysaccharide.

1.2.8 Pneumococcal Polysaccharide Serotype 33F

As shown at FIG. 7, the polysaccharide repeating unit of serotype 33Fconsists of a branched pentasaccharide backbone (two galactopyranoses(Gal_(p)), two galactofuranoses (Gal_(f)) and one glucopyranose(Glc_(p)) with a terminal αGal_(p) linked to the C2 hydroxyl group ofαGal_(p) residue within the backbone (Lemercinier et al. (2006)Carbohydrate Research 341(1):68-74.). It has been reported in theliterature that the C2 hydroxyl group of the backbone 3-β-Gal_(f)residue is O-acetylated.

Serotype 33F polysaccharides can be obtained directly from bacteriausing isolation procedures known to one of ordinary skill in the art(see for example methods disclosed in U.S. Patent App. Pub. Nos.2006/0228380, 2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340,and 2008/0102498 and WO 2008/118752). In addition, they can be producedusing synthetic protocols.

Serotype 33F S. pneumoniae strains may be obtained from establishedculture collections (such as for example the Streptococcal ReferenceLaboratory (Centers for Disease Control and Prevention, Atlanta, Ga.))or clinical specimens.

Purified polysaccharides from serotype 33F may be activated (e.g.,chemically activated) to make them capable of reacting and thenincorporated into glycoconjugates of the invention, as further describedherein.

The isolated serotype 33F capsular polysaccharide obtained bypurification of serotype 33F polysaccharide from the S. pneumoniaelysate and optionally sizing of the purified polysaccharide can becharacterized by different parameters including, for example, themolecular weight and the mM of acetate per mM of said serotype 33Fcapsular polysaccharide.

In some embodiments, the purified polysaccharides from S. pneumoniaeserotype 33F before conjugation have a molecular weight of betweenbetween 10 kDa and 2,000 kDa. In other such embodiments, the saccharidehas a molecular weight of between 50 kDa and 2,000 kDa. In further suchembodiments, the saccharide has a molecular weight of between 50 kDa and1,750 kDa; between 50 kDa and 1,500 kDa; between 50 kDa and 1,250 kDa;between 50 kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDaand 500 kDa; between 100 kDa and 2,000 kDa; between 100 kDa and 1,750kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa;between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100kDa and 500 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa;between 200 kDa and 1,000 kDa; between 200 kDa and 750 kDa; or between200 kDa and 500 kDa. Any whole number integer within any of the aboveranges is contemplated as an embodiment of the disclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described herein,polysaccharide can be subjected to sizing techniques before conjugation.The molecular weight ranges mentioned above refer to purifiedpolysaccharides before conjugation (e.g., before activation) after aneventual sizing step.

The presence of O-acetyl in a purified, isolated or activated serotype33F capsular polysaccharide or in a serotype 33F polysaccharide-carrierprotein conjugate is expressed as the number of mM of acetate per mM ofsaid polysaccharide or as the number of O-acetyl group perpolysaccharide repeating unit.

In a preferred embodiment, the purified polysaccharides from S.pneumoniae serotype 33F has at least 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4or 1.6, μmol acetate per μmol of said serotype 33F capsularpolysaccharide.

1.3 Glycoconjugates of the Invention

The purified saccharides are chemically activated to make thesaccharides (i.e., activated saccharides) capable of reacting with thecarrier protein. Once activated, each capsular saccharide is separatelyconjugated to a carrier protein to form a glycoconjugate. In oneembodiment, each capsular saccharide is conjugated to the same carrierprotein. The chemical activation of the saccharides and subsequentconjugation to the carrier protein can be achieved by the activation andconjugation methods disclosed herein.

1.3.1 Glycoconjugates from S. pneumoniae Serotype 1, 3, 4, 5, 6A, 6B,7F, 9V, 14, 18C, 19A, 19F and 23F

Capsular polysaccharides from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F of S. pneumoniae are prepared by standardtechniques known to those of ordinary skill in the art (see for exampleWO 2006/110381, WO 2008/118752, WO 2006/110352, and U.S. Patent App.Pub. Nos. 2006/0228380, 2006/0228381, 2008/0102498 and 2008/0286838).

In an embodiment, the polysaccharides are activated with1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form acyanate ester. The activated polysaccharide is then coupled directly orvia a spacer (linker) group to an amino group on the carrier protein(preferably CRM₁₉₇). For example, the spacer could be cystamine orcysteamine to give a thiolated polysaccharide which could be coupled tothe carrier via a thioether linkage obtained after reaction with amaleimide-activated carrier protein (for example usingN-[γ-maleimidobutyrloxy]succinimide ester (GMBS)) or a haloacetylatedcarrier protein (for example using iodoacetimide, N-succinimidylbromoacetate (SBA; SIB), N-succinimidyl(4-iodoacetyl)aminobenzoate(SIAB), sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo-SIAB),N-succinimidyl iodoacetate (SIA) or succinimidyl3-[bromoacetamido]proprionate (SBAP)). Preferably, the cyanate ester(optionally made by CDAP chemistry) is coupled with hexane diamine oradipic acid dihydrazide (ADH) and the amino-derivatised saccharide isconjugated to the carrier protein (e.g., CRM₁₉₇) using carbodiimide(e.g., EDAC or EDC) chemistry via a carboxyl group on the proteincarrier. Such conjugates are described for example in WO 93/15760, WO95/08348 and WO 96/129094.

Other suitable techniques for conjugation use carbodiimides, hydrazides,active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide,S--NHS, EDC, TSTU. Many are described in International PatentApplication Publication No. WO 98/42721. Conjugation may involve acarbonyl linker which may be formed by reaction of a free hydroxyl groupof the saccharide with 1,1′-carbonyldiimidazole (CDI) (see Bethell etal. (1979) J. Biol. Chem. 254:2572-2574; Hearn et al. (1981) J.Chromatogr. 218:509-518) followed by reaction with a protein to form acarbamate linkage. This may involve reduction of the anomeric terminusto a primary hydroxyl group, optional protection/deprotection of theprimary hydroxyl group, reaction of the primary hydroxyl group with CDIto form a CDI carbamate intermediate and coupling the CDI carbamateintermediate with an amino group on a protein.

In an preferred embodiment, at least one of capsular polysaccharidesfrom serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F ofS. pneumoniae is conjugated to the carrier protein by reductiveamination (such as described in U.S. Patent Appl. Pub. Nos.2006/0228380, 2007/0231340, 2007/0184071 and 2007/0184072, WO2006/110381, WO 2008/079653, and WO 2008/143709). In a preferredembodiment, the capsular polysaccharides from serotypes 1, 3, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19A, 19F and 23F of S. pneumoniae are allconjugated to the carrier protein by reductive amination.

Reductive amination involves two steps: (1) oxidation of thepolysaccharide and (2) reduction of the activated polysaccharide and acarrier protein to form a conjugate. Before oxidation, thepolysaccharide is optionally hydrolyzed. Mechanical or chemicalhydrolysis may be employed. Chemical hydrolysis may be conducted usingacetic acid. The oxidation step may involve reaction with periodate. Forthe purpose of the present invention, the term “periodate” includes bothperiodate and periodic acid; the term also includes both metaperiodate(IO₄ ⁻) and orthoperiodate (IO₆ ⁵⁻) and the various salts of periodate(e.g., sodium periodate and potassium periodate).

In an embodiment the capsular polysaccharide from serotype 1, 3, 4, 5,6A, 6B, 7F, 9V, 14, 18C, 19A, 19F or 23F of S. pneumoniae is oxidized inthe presence of metaperiodate, preferably in the presence of sodiumperiodate (NaIO₄). In another embodiment the capsular polysaccharidesfrom serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F ofS. pneumoniae is oxydized in the presence of orthoperiodate, preferablyin the presence of periodic acid.

Following the oxidation step of the polysaccharide, the polysaccharideis said to be activated and is referred to as “activated polysaccharide”here below. The activated polysaccharide and the carrier protein may belyophilised (freeze-dried), either independently (discretelyophilization) or together (co-lyophilized). In one embodiment theactivated polysaccharide and the carrier protein are co-lyophilized. Inanother embodiment the activated polysaccharide and the carrier proteinare lyophilized independently.

In one embodiment the lyophilization takes place in the presence of anon-reducing sugar, possible non-reducing sugars include sucrose,trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitoland palatinit.

The second step of the conjugation process is the reduction of theactivated polysaccharide and a carrier protein to form a conjugate(so-called reductive amination), using a reducing agent. Reducing agentswhich are suitable include the cyanoborohydrides, such as sodiumcyanoborohydride, borane-pyridine, or borohydride exchange resin. In oneembodiment the reducing agent is sodium cyanoborohydride.

In an embodiment, the reduction reaction is carried out in aqueoussolvent, in another embodiment the reaction is carried out in aproticsolvent. In an embodiment, the reduction reaction is carried out in DMSO(dimethylsulfoxide) or in DMF (dimethylformamide) solvent. The DMSO orDMF solvent may be used to reconstitute the activated polysaccharide andcarrier protein which has been lyophilized.

At the end of the reduction reaction, there may be unreacted aldehydegroups remaining in the conjugates, these may be capped using a suitablecapping agent. In one embodiment this capping agent is sodiumborohydride (NaBH₄). Following the conjugation (the reduction reactionand optionally the capping), the glycoconjugates may be purified. Theglycoconjugates maybe purified by diafiltration and/or ion exchangechromatography and/or size exclusion chromatography. In an embodiment,the glycoconjugates are purified by diafiltration or ion exchangechromatography or size exclusion chromatography. In one embodiment theglycoconjugates are sterile filtered.

In some embodiments, the glycoconjugate from S. pneumoniae serotypes 9Vand/or 18C comprise a saccharide which has a degree of O-acetylation ofbetween 10% and 100%, between 20% and 100%, between 30% and 100%,between 40% and 100%, between 50% and 100%, between 60% and 100%,between 70% and 100%, between 75% and 100%, between 80% and 100%,between 90% and 100%, between 50% and 90%, between 60% and 90%, between70% and 90% or between 80% and 90%. In other embodiments, the degree ofO-acetylation is ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, or≥90%, or about 100%.

In some embodiments, the glycoconjugate from S. pneumoniae serotypes 9Vand/or 18C of the invention are O-acetylated. In some embodiments, theglycoconjugate from S. pneumoniae serotype 9V is O-acetylated and theglycoconjugate from S. pneumoniae serotype 18C is de-O-acetylated.

1.3.2 Glycoconjugates from S. pneumoniae Serotype 22F

In an embodiment, the serotype 22F glycoconjugates are obtained byactivating polysaccharide with 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate (CDAP) to form a cyanate ester. The activatedpolysaccharide may be coupled directly or via a spacer (linker) group toan amino group on the carrier protein. For example, the spacer could becystamine or cysteamine to give a thiolated polysaccharide which couldbe coupled to the carrier via a thioether linkage obtained afterreaction with a maleimide-activated carrier protein (for example usingGMBS) or a haloacetylated carrier protein (for example usingiodoacetimide, SIB, SIAB, sulfo-SIAB, SIA, or SBAP). Preferably, thecyanate ester (optionally made by CDAP chemistry) is coupled with hexanediamine or adipic acid dihydrazide (ADH) and the amino-derivatisedsaccharide is conjugated to the carrier protein using carbodiimide(e.g., EDAC or EDC) chemistry via a carboxyl group on the proteincarrier. Such conjugates are described for example in WO 93/15760, WO95/08348 and WO 96/129094.

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S--NHS, EDC, TSTU.Many are described in International Patent Application Publication No.WO 98/42721. Conjugation may involve a carbonyl linker which may beformed by reaction of a free hydroxyl group of the saccharide with CDI(see Bethell et al. (1979) J. Biol. Chem. 254:2572-2574; Hearn et al.(1981) J. Chromatogr. 218:509-518) followed by reaction with a proteinto form a carbamate linkage. This may involve reduction of the anomericterminus to a primary hydroxyl group, optional protection/deprotectionof the primary hydroxyl group, reaction of the primary hydroxyl groupwith CDI to form a CDI carbamate intermediate and coupling the CDIcarbamate intermediate with an amino group on a protein.

In preferred embodiments, the serotype 22F glycoconjugates of theinvention are prepared using reductive amination. Reductive aminationinvolves two steps: (1) oxidation of the polysaccharide to generatealdehyde functionalities from vicinal diols in individual hexasaccharideunit and (2) reduction of the activated polysaccharide and a carrierprotein (e.g., CRM₁₉₇) to form a conjugate.

Preferably, before oxidation, sizing of the serotype 22F polysaccharideto a target molecular weight (MW) range is performed. Advantageously,the size of the purified serotype 22F polysaccharide is reduced whilepreserving critical features of the structure of the polysaccharide suchas for example the presence of O-acetyl groups.

Preferably, the size of the purified serotype 22F polysaccharide isreduced by mechanical homogenization (see section 1.2.7 above).

In an embodiment, serotype polysaccharide is activated (oxidized) by aprocess comprising the step of:

(a) reacting isolated serotype 22F polysaccharide with an oxidizingagent; and(b) quenching the oxidation reaction by addition of a quenching agentresulting in an activated serotype 22F polysaccharide.

In a preferred embodiment, the oxidizing agent is periodate. For thepurpose of the present invention, the term “periodate” includes bothperiodate and periodic acid; the term also includes both metaperiodate(IO₄ ⁻) and orthoperiodate (IO₆ ⁵⁻) and the various salts of periodate(e.g., sodium periodate and potassium periodate). In a preferredembodiment, the oxidizing agent is sodium periodate. In a preferredembodiment, the periodate used for the oxidation of serotype 22Fpolysaccharide is metaperiodate. In a preferred embodiment the periodateused for the oxidation of serotype 22F polysaccharide is sodiummetaperiodate.

In one embodiment, the quenching agent is selected from vicinal diols,1,2-aminoalcohols, amino acids, glutathione, sulfite, bisulfate,dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites orphosphorous acid.

In one embodiment, the quenching agent is a 1,2-aminoalcohols of formula(I):

wherein R¹ is selected from H, methyl, ethyl, propyl or isopropyl.

In one embodiment, the quenching agent is selected from sodium andpotassium salts of sulfite, bisulfate, dithionite, metabisulfite,thiosulfate, phosphites, hypophosphites or phosphorous acid.

In one embodiment, the quenching agent is an amino acid. In suchembodiments, said amino acid may be selected from serine, threonine,cysteine, cystine, methionine, proline, hydroxyproline, tryptophan,tyrosine, and histidine.

In one embodiment, the quenching agent is a sulfite such as bisulfate,dithionite, metabisulfite, thiosulfate.

In one embodiment, the quenching agent is a compound comprising twovicinal hydroxyl groups (vicinal diols), i.e., two hydroxyl groupscovalently linked to two adjacent carbon atoms.

Preferably, the quenching agent is a compound of formula (II):

wherein R¹ and R² are each independently selected from H, methyl, ethyl,propyl or isopropyl.

In a preferred embodiment, the quenching agent is glycerol, ethyleneglycol, propan-1,2-diol, butan-1,2-diol or butan-2,3-diol, or ascorbicacid. In a preferred embodiment, the quenching agent is butan-2,3-diol.

In a preferred embodiment, the isolated serotype 22F polysaccharide isactivated by a process comprising the step of:

(a) reacting isolated serotype 22F polysaccharide with periodate; and(b) quenching the oxidation reaction by addition of butan-2,3-diolresulting in an activated serotype 22F polysaccharide.

Following the oxidation step of the polysaccharide, the polysaccharideis said to be activated and is referred to as “activated polysaccharide”here below.

In a preferred embodiment, the activated serotype 22F polysaccharide ispurified. The activated serotype 22F polysaccharide is purifiedaccording to methods known to the man skilled in the art such as gelpermeation chromatography (GPC), dialysis orultrafiltration/diafiltration. For example, the activated 22Fpolysaccharide is purified by concentration and diafiltration using anultrafiltration device.

In a preferred embodiment the degree of oxidation of the activatedserotype 22F polysaccharide is between 2 and 30, between 2 and 25,between 2 and 20, between 2 and 15, between 2 and 10, between 2 and 5,between 5 and 30, between 5 and 25, between 5 and 20, between 5 and 15,between 5 and 10, between 10 and 30, between 10 and 25, between 10 and20, between 10 and 15, between 15 and 30, between 15 and 25, between 15and 20, between 20 to 30, or between 20 to 25. In a preferred embodimentthe degree of oxidation of the activated serotype 22F polysaccharide isbetween 2 and 10, between 4 and 8, between 4 and 6, between 6 and 8,between 6 and 12, between 8 and 14, between 9 and 11, between 10 and 16,between 12 and 16, between 14 and 18, between 16 and 20, between 16 and18, between 18 and 22, or between 18 and 20.

In a preferred embodiment, the activated serotype 22F polysaccharide hasa molecular weight between 25 kDa and 1,000 kDa, between 100 kDa and1,000 kDa, between 300 kDa and 800 kDa, between 300 kDa and 700 kDa,between 300 kDa and 600 kDa, between 400 kDa and 1,000 kDa, between 400kDa and 800 kDa, between 400 kDa and 700 kDa or between 400 kDa and 600kDa. In an embodiment, the activated serotype 22F polysaccharide has amolecular weight between 300 kDa and 800 kDa. In an embodiment, theactivated serotype 22F polysaccharide has a molecular weight between 400kDa and 600 kDa. In a preferred embodiment, the activated serotype 22Fpolysaccharide has a molecular weight between 400 kda and 600 kDa and adegree of oxidation between 10 and 25, between 10 and 20, between 12 and20 or between 14 and 18. In a preferred embodiment, the activatedserotype 22F polysaccharide has a molecular weight between 400 kDa and600 kDa and a degree of oxidation between 10 and 20.

In a preferred embodiment, the activated serotype 22F polysaccharidecomprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7 or about 0.8 mMacetate per mM serotype 22F polysaccharide. In a preferred embodiment,the activated serotype 22F polysaccharide comprises at least 0.5, 0.6 or0.7 mM acetate per mM serotype 22F polysaccharide. In a preferredembodiment, the activated serotype 22F polysaccharide comprises at least0.6 mM acetate per mM serotype 22F polysaccharide. In a preferredembodiment, the activated serotype 22F polysaccharide comprises at least0.7 mM acetate per mM serotype 22F polysaccharide.

In a preferred embodiment, the activated serotype 22F polysaccharide hasa molecular weight between 400 kDa and 800 kDa and comprises at least0.6 mM acetate per mM serotype 22F polysaccharide.

In a preferred embodiment, the activated serotype 22F polysaccharide hasa molecular weight between 400 kDa and 800 kDa, a degree of oxidationbetween 12 and 20 and comprises at least 0.6 mM acetate per mM serotype22F polysaccharide.

The activated polysaccharide and/or the carrier protein may belyophilised (freeze-dried), either independently (discretelyophilization) or together (co-lyophilized).

In an embodiment, the activated serotype 22F polysaccharide islyophilized, optionally in the presence of saccharide. In a preferredembodiment, the saccharide is selected from sucrose, trehalose,raffinose, stachyose, melezitose, dextran, mannitol, lactitol andpalatinit. In a preferred embodiment, the saccharide is sucrose. In oneembodiment, the lyophilized activated polysaccharide is then compoundedwith a solution comprising the carrier protein.

In another embodiment the activated polysaccharide and the carrierprotein are co-lyophilised. In such embodiments, the activated serotype22F polysaccharide is compounded with the carrier protein andlyophilized optionally in the presence of a saccharide. In a preferredembodiment, the saccharide is selected from sucrose, trehalose,raffinose, stachyose, melezitose, dextran, mannitol, lactitol andpalatinit. In a preferred embodiment, the saccharide is sucrose. Theco-lyophilized polysaccharide and carrier protein can then beresuspended in solution and reacted with a reducing agent.

The second step of the conjugation process is the reduction of theactivated polysaccharide and a carrier protein to form a conjugate(reductive amination), using a reducing agent.

The activated serotype 22F polysaccharide can be conjugated to a carrierprotein by a process comprising the step of:

(c) compounding the activated serotype 22F polysaccharide with a carrierprotein; and(d) reacting the compounded activated serotype 22F polysaccharide andcarrier protein with a reducing agent to form a serotype 22Fpolysaccharide-carrier protein conjugate.

In an embodiment, the reduction reaction is carried out in aqueoussolvent. In another embodiment the reaction is carried out in aproticsolvent. In an embodiment, the reduction reaction is carried out in DMSO(dimethylsulfoxide) or in DMF (dimethylformamide)) solvent. The DMSO orDMF solvent may be used to reconstitute the activated polysaccharide andcarrier protein which has been lyophilised.

The conjugation of activated serotype 22F polysaccharide with a proteincarrier by reductive amination in dimethylsulfoxide (DMSO) is suitableto preserve the O-acetyl content of the polysaccharide as compared, forexample, to reductive amination in aqueous phase where the level ofO-acetylation of the polysaccharide may be significantly reduced.Therefore in a preferred embodiment, step (c) and step (d) are carriedout in DMSO.

In an embodiment, the reducing agent is sodium cyanoborohydride, sodiumtriacetoxyborohydride, sodium or zinc borohydride in the presence ofBronsted or Lewis acids, amine boranes such as pyridine borane,2-Picoline Borane, 2,6-diborane-methanol, dimethylamine-borane,t-BuMe^(i)PrN—BH₃, benzylamine-BH₃ or 5-ethyl-2-methylpyridine borane(PEMB). In a preferred embodiment, the reducing agent is sodiumcyanoborohydride.

At the end of the reduction reaction, there may be unreacted aldehydegroups remaining in the conjugates, these may be capped using a suitablecapping agent. In one embodiment this capping agent is sodiumborohydride (NaBH₄).

Following conjugation of serotype 22F polysaccharide to the carrierprotein, the glycoconjugate can be purified (enriched with respect tothe amount of polysaccharide-protein conjugate) by a variety oftechniques known to the skilled person. These techniques includedialysis, concentration/diafiltration operations, tangential flowfiltration precipitation/elution, column chromatography (DEAE orhydrophobic interaction chromatography), and depth filtration.

In some embodiments, the serotype 22F glycoconjugates of the presentinvention comprise a saccharide having a molecular weight of between 10kDa and 2,000 kDa. In other such embodiments, the saccharide has amolecular weight of between 50 kDa and 1,000 kDa. In other suchembodiments, the saccharide has a molecular weight of between 70 kDa and900 kDa. In other such embodiments, the saccharide has a molecularweight of between 100 kDa and 800 kDa. In other such embodiments, thesaccharide has a molecular weight of between 200 kDa and 600 kDa. Infurther such embodiments, the saccharide has a molecular weight of 100kDa to 1,000 kDa; 100 kDa to 900 kDa; 100 kDa to 800 kDa; 100 kDa to 700kDa; 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa; 100 kDato 300 kDa; 150 kDa to 1,000 kDa; 150 kDa to 900 kDa; 150 kDa to 800kDa; 150 kDa to 700 kDa; 150 kDa to 600 kDa; 150 kDa to 500 kDa; 150 kDato 400 kDa; 150 kDa to 300 kDa; 200 kDa to 1,000 kDa; 200 kDa to 900kDa; 200 kDa to 800 kDa; 200 kDa to 700 kDa; 200 kDa to 600 kDa; 200 kDato 500 kDa; 200 kDa to 400 kDa; 200 kDa to 300 kDa; 250 kDa to 1,000kDa; 250 kDa to 900 kDa; 250 kDa to 800 kDa; 250 kDa to 700 kDa; 250 kDato 600 kDa; 250 kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa;300 kDa to 1000 kDa; 300 kDa to 900 kDa; 300 kDa to 800 kDa; 300 kDa to700 kDa; 300 kDa to 600 kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400kDa to 1,000 kDa; 400 kDa to 900 kDa; 400 kDa to 800 kDa; 400 kDa to 700kDa; 400 kDa to 600 kDa; 500 kDa to 600 kDa. Any whole number integerwithin any of the above ranges is contemplated as an embodiment of thedisclosure. In some such embodiments, the serotype 22F glycoconjugatesare prepared using reductive amination.

In some embodiments, the serotype 22F glycoconjugate of the inventionhas a molecular weight of between 400 kDa and 15,000 kDa; between 500kDa and 10,000 kDa; between 2,000 kDa and 10,000 kDa; between 3,000 kDaand 8,000 kDa; or between 3,000 kDa and 5,000 kDa. In other embodiments,the serotype 22F glycoconjugate has a molecular weight of between 500kDa and 10,000 kDa. In other embodiments, the serotype 22Fglycoconjugate has a molecular weight of between 1,000 kDa and 8,000kDa. In still other embodiments, the serotype 22F glycoconjugate has amolecular weight of between 2,000 kDa and 8,000 kDa or between 3,000 kDaand 7,000 kDa. In further embodiments, the serotype 22F glycoconjugateof the invention has a molecular weight of between 200 kDa and 20,000kDa; between 200 kDa and 15,000 kDa; between 200 kDa and 10,000 kDa;between 200 kDa and 7,500 kDa; between 200 kDa and 5,000 kDa; between200 kDa and 3,000 kDa; between 200 kDa and 1,000 kDa; between 500 kDaand 20,000 kDa; between 500 kDa and 15,000 kDa; between 500 kDa and12,500 kDa; between 500 kDa and 10,000 kDa; between 500 kDa and 7,500kDa; between 500 kDa and 6,000 kDa; between 500 kDa and 5,000 kDa;between 500 kDa and 4,000 kDa; between 500 kDa and 3,000 kDa; between500 kDa and 2,000 kDa; between 500 kDa and 1,500 kDa; between 500 kDaand 1,000 kDa; between 750 kDa and 20,000 kDa; between 750 kDa and15,000 kDa; between 750 kDa and 12,500 kDa; between 750 kDa and 10,000kDa; between 750 kDa and 7,500 kDa; between 750 kDa and 6,000 kDa;between 750 kDa and 5,000 kDa; between 750 kDa and 4,000 kDa; between750 kDa and 3,000 kDa; between 750 kDa and 2,000 kDa; between 750 kDaand 1,500 kDa; between 1,000 kDa and 15,000 kDa; between 1,000 kDa and12,500 kDa; between 1,000 kDa and 10,000 kDa; between 1,000 kDa and7,500 kDa; between 1,000 kDa and 6,000 kDa; between 1,000 kDa and 5,000kDa; between 1,000 kDa and 4,000 kDa; between 1,000 kDa and 2,500 kDa;between 2,000 kDa and 15,000 kDa; between 2,000 kDa and 12,500 kDa;between 2,000 kDa and 10,000 kDa; between 2,000 kDa and 7,500 kDa;between 2,000 kDa and 6,000 kDa; between 2,000 kDa and 5,000 kDa;between 2,000 kDa and 4,000 kDa; or between 2,000 kDa and 3,000 kDa.

In further embodiments, the serotype 22F glycoconjugate of the inventionhas a molecular weight of between 3,000 kDa and 20,000 kDa; between3,000 kDa and 15,000 kDa; between 3,000 kDa and 10,000 kDa; between3,000 kDa and 7,500 kDa; between 3,000 kDa and 5,000 kDa; between 4,000kDa and 20,000 kDa; between 4,000 kDa and 15,000 kDa; between 4,000 kDaand 12,500 kDa; between 4,000 kDa and 10,000 kDa; between 4,000 kDa and7,500 kDa; between 4,000 kDa and 6,000 kDa; or between 4,000 kDa and5,000 kDa.

In further embodiments, the serotype 22F glycoconjugate of the inventionhas a molecular weight of between 5,000 kDa and 20,000 kDa; between5,000 kDa and 15,000 kDa; between 5,000 kDa and 10,000 kDa; between5,000 kDa and 7,500 kDa; between 6,000 kDa and 20,000 kDa; between 6,000kDa and 15,000 kDa; between 6,000 kDa and 12,500 kDa; between 6,000 kDaand 10,000 kDa or between 6,000 kDa and 7,500 kDa.

The molecular weight of the glycoconjugate is measured by SEC-MALLS. Anywhole number integer within any of the above ranges is contemplated asan embodiment of the disclosure.

In a preferred embodiment, the serotype 22F glycoconjugate of theinvention comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7 orabout 0.8 mM acetate per mM serotype 22F polysaccharide. In a preferredembodiment, the glycoconjugate comprises at least 0.5, 0.6 or 0.7 mMacetate per mM serotype 22F polysaccharide. In a preferred embodiment,the glycoconjugate comprises at least 0.6 mM acetate per mM serotype 22Fpolysaccharide. In a preferred embodiment, the glycoconjugate comprisesat least 0.7 mM acetate per mM serotype 22F polysaccharide.

In a preferred embodiment, the ratio of mM acetate per mM serotype 22Fpolysaccharide in the glycoconjugate to mM acetate per mM serotype 22Fpolysaccharide in the isolated polysaccharide is at least 0.6, 0.65,0.7, 0.75, 0.8, 0.85, 0.9, or 0.95. In a preferred embodiment, the ratioof mM acetate per mM serotype 22F polysaccharide in the glycoconjugateto mM acetate per mM serotype 22F polysaccharide in the isolatedpolysaccharide is at least 0.7. In a preferred embodiment, the ratio ofmM acetate per mM serotype 22F polysaccharide in the glycoconjugate tomM acetate per mM serotype 22F polysaccharide in the isolatedpolysaccharide is at least 0.9.

In a preferred embodiment, the ratio of mM acetate per mM serotype 22Fpolysaccharide in the glycoconjugate to mM acetate per mM serotype 22Fpolysaccharide in the activated polysaccharide is at least 0.6, 0.65,0.7, 0.75, 0.8, 0.85, 0.9, or 0.95. In a preferred embodiment, the ratioof mM acetate per mM serotype 22F polysaccharide in the glycoconjugateto mM acetate per mM serotype 22F polysaccharide in the activatedpolysaccharide is at least 0.7. In a preferred embodiment, the ratio ofmM acetate per mM serotype 22F polysaccharide in the glycoconjugate tomM acetate per mM serotype 22F polysaccharide in the activatedpolysaccharide is at least 0.9.

Another way to characterize the serotype 22F glycoconjugates of theinvention is by the number of lysine residues in the carrier protein(e.g., CRM₁₉₇) that become conjugated to the saccharide which can becharacterized as a range of conjugated lysines (degree of conjugation).The evidence for lysine modification of the carrier protein, due tocovalent linkages to the polysaccharides, can be obtained by amino acidanalysis using routine methods known to those of skill in the art.Conjugation results in a reduction in the number of lysine residuesrecovered compared to the CRM₁₉₇ protein starting material used togenerate the conjugate materials. In a preferred embodiment, the degreeof conjugation of the serotype 22F glycoconjugate of the invention isbetween 2 and 15, between 2 and 13, between 2 and 10, between 2 and 8,between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 15,between 3 and 13, between 3 and 10, between 3 and 8, between 3 and 6,between 3 and 5, between 3 and 4, between 5 and 15, between 5 and 10,between 8 and 15, between 8 and 12, between 10 and 15 or between 10 and12. In an embodiment, the degree of conjugation of the serotype 22Fglycoconjugate of the invention is about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14 or about 15. In a preferred embodiment, the degree ofconjugation of the serotype 22F glycoconjugate of the invention isbetween 4 and 7. In some such embodiments, the carrier protein isCRM₁₉₇.

The serotype 22F glycoconjugates of the invention may also becharacterized by the ratio (weight/weight) of saccharide to carrierprotein. In some embodiments, the ratio of serotype 22F polysaccharideto carrier protein in the glycoconjugate (w/w) is between 0.5 and 3.0(e.g., about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0,about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3,about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, orabout 3.0). In other embodiments, the saccharide to carrier proteinratio (w/w) is between 0.5 and 2.0, between 0.5 and 1.5, between 0.8 and1.2, between 0.5 and 1.0, between 1.0 and 1.5 or between 1.0 and 2.0. Infurther embodiments, the saccharide to carrier protein ratio (w/w) isbetween 0.8 and 1.2. In a preferred embodiment, the ratio of serotype22F capsular polysaccharide to carrier protein in the conjugate isbetween 0.9 and 1.1. In some such embodiments, the carrier protein isCRM₁₉₇.

The serotype 22F glycoconjugates and immunogenic compositions of theinvention may contain free saccharide that is not covalently conjugatedto the carrier protein, but is nevertheless present in theglycoconjugate composition. The free saccharide may be noncovalentlyassociated with (i.e., noncovalently bound to, adsorbed to, or entrappedin or with) the glycoconjugate.

In a preferred embodiment, the serotype 22F glycoconjugate comprisesless than about 50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% of freeserotype 22F polysaccharide compared to the total amount of serotype 22Fpolysaccharide. In a preferred embodiment the serotype 22Fglycoconjugate comprises less than about 40% of free serotype 22Fpolysaccharide compared to the total amount of serotype 22Fpolysaccharide. In a preferred embodiment the serotype 22Fglycoconjugate comprises less than about 25% of free serotype 22Fpolysaccharide compared to the total amount of serotype 22Fpolysaccharide. In a preferred embodiment the serotype 22Fglycoconjugate comprises less than about 20% of free serotype 22Fpolysaccharide compared to the total amount of serotype 22Fpolysaccharide. In a preferred embodiment the serotype 22Fglycoconjugate comprises less than about 15% of free serotype 22Fpolysaccharide compared to the total amount of serotype 22Fpolysaccharide.

The serotype 22F glycoconjugates may also be characterized by theirmolecular size distribution (K_(d)). Size exclusion chromatography media(CL-4B) can be used to determine the relative molecular sizedistribution of the conjugate. Size Exclusion Chromatography (SEC) isused in gravity fed columns to profile the molecular size distributionof conjugates. Large molecules excluded from the pores in the mediaelute more quickly than small molecules. Fraction collectors are used tocollect the column eluate. The fractions are tested colorimetrically bysaccharide assay. For the determination of K_(d), columns are calibratedto establish the fraction at which molecules are fully excluded (V₀),(K_(d)=0), and the fraction representing the maximum retention (V_(i)),(K_(d)=1). The fraction at which a specified sample attribute is reached(V_(e)), is related to K_(d) by the expression,K_(d)=(V_(e)−V₀)/(V_(i)−V₀).

In a preferred embodiment, at least 30% of the serotype 22Fglycoconjugate has a K_(d) below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 40% of the glycoconjugate has a K_(d)below or equal to 0.3 in a CL-4B column. In a preferred embodiment, atleast 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the serotype 22Fglycoconjugate has a K_(d) below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 60% of the serotype 22F glycoconjugatehas a K_(d) below or equal to 0.3 in a CL-4B column. In a preferredembodiment, between 50% and 80% of the serotype 22F glycoconjugate has aK_(d) below or equal to 0.3 in a CL-4B column. In a preferredembodiment, between 65% and 80% of the serotype 22F glycoconjugate has aK_(d) below or equal to 0.3 in a CL-4B column.

1.3.3 Glycoconjugates from S. pneumoniae Serotype 33F

In an embodiment, the serotype 33F glycoconjugates are obtained byactivating polysaccharide with 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate (CDAP) to form a cyanate ester. The activatedpolysaccharide may be coupled directly or via a spacer (linker) group toan amino group on the carrier protein. For example, the spacer could becystamine or cysteamine to give a thiolated polysaccharide which couldbe coupled to the carrier via a thioether linkage obtained afterreaction with a maleimide-activated carrier protein (for example usingGMBS) or a haloacetylated carrier protein (for example usingiodoacetimide, SIB, SIAB, sulfo-SIAB, SIA, or SBAP). Preferably, thecyanate ester (optionally made by CDAP chemistry) is coupled with hexanediamine or adipic acid dihydrazide (ADH) and the amino-derivatisedsaccharide is conjugated to the carrier protein using carbodiimide(e.g., EDAC or EDC) chemistry via a carboxyl group on the proteincarrier. Such conjugates are described for example in WO 93/15760, WO95/08348 and WO 96/129094.

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S--NHS, EDC, TSTU.Many are described in International Patent Application Publication No.WO 98/42721. Conjugation may involve a carbonyl linker which may beformed by reaction of a free hydroxyl group of the saccharide with CDI(see Bethell et al. (1979) J. Biol. Chem. 254:2572-2574; Hearn et al.(1981) J. Chromatogr. 218:509-518) followed by reaction with a proteinto form a carbamate linkage. This may involve reduction of the anomericterminus to a primary hydroxyl group, optional protection/deprotectionof the primary hydroxyl group, reaction of the primary hydroxyl groupwith CDI to form a CDI carbamate intermediate and coupling the CDIcarbamate intermediate with an amino group on a protein.

In certain embodiments, the serotype 33F glycoconjugates of theinvention are prepared using reductive amination. In such embodiment,the serotype 33F glycoconjugates of the invention maybe prepared usingreductive amination in aqueous phase (RAC/aqueous). Reductive aminationin aqueous phase has been successfully applied to produce pneumococcalconjugate vaccine (see, e.g., WO 2006/110381). Preferably though, whenusing reductive amination, the serotype 33F glycoconjugates are preparedvia reductive amination in DMSO (RAC/DMSO). In view of the challengesassociated with the preservation of O-acetyl functionality usingRAC/aqueous process, reductive amination in DMSO is preferred. RAC/DMSOhas been successfully applied to produce pneumococcal conjugate vaccine(see, e.g., WO 2006/110381).

In preferred embodiments, the serotype 33F glycoconjugates of theinvention are prepared using eTEC conjugation (hereinafter “serotype 33FeTEC linked glycoconjugates”), such as described at Examples 1, 2 and 3and in WO 2014/027302. Said 33F glycoconjugates comprise a saccharidecovalently conjugated to a carrier protein through one or more eTECspacers, wherein the saccharide is covalently conjugated to the eTECspacer through a carbamate linkage, and wherein the carrier protein iscovalently conjugated to the eTEC spacer through an amide linkage. TheeTEC linked glycoconjugates of the invention may be represented by thegeneral formula (111):

wherein the atoms that comprise the eTEC spacer are contained in thecentral box.

The eTEC spacer includes seven linear atoms (i.e.,—C(O)NH(CH₂)₂SCH₂C(O)—) and provides stable thioether and amide bondsbetween the saccharide and carrier protein. Synthesis of the eTEC linkedglycoconjugate involves reaction of an activated hydroxyl group of thesaccharide with the amino group of a thioalkylamine reagent, e.g.,cystamine or cysteinamine or a salt thereof, forming a carbamate linkageto the saccharide to provide a thiolated saccharide. Generation of oneor more free sulfhydryl groups is accomplished by reaction with areducing agent to provide an activated thiolated saccharide. Reaction ofthe free sulfhydryl groups of the activated thiolated saccharide with anactivated carrier protein having one or more α-haloacetamide groups onamine containing residues generates a thioether bond to form theconjugate, wherein the carrier protein is attached to the eTEC spacerthrough an amide bond.

In serotype 33F glycoconjugates of the invention, the saccharide may bea polysaccharide or an oligosaccharide. The carrier protein may beselected from any suitable carrier as described herein or known to thoseof skill in the art. In frequent embodiments, the saccharide is apolysaccharide. In some such embodiments, the carrier protein is CRM₁₉₇.In some such embodiments, the eTEC linked glycoconjugate comprises a S.pneumoniae serotype 33F capsular polysaccharide.

In particularly preferred embodiments, the eTEC linked glycoconjugatecomprises a Pn-33F capsular polysaccharide, which is covalentlyconjugated to CRM₁₉₇ through an eTEC spacer (serotype 33F eTEC linkedglycoconjugates).

In some embodiments, the serotype 33F glycoconjugates of the presentinvention comprise a saccharide having a molecular weight of between 10kDa and 2,000 kDa. In other such embodiments, the saccharide has amolecular weight of between 50 kDa and 2,000 kDa. In further suchembodiments, the saccharide has a molecular weight of between 50 kDa and1,750 kDa; between 50 kDa and 1,500 kDa; between 50 kDa and 1,250 kDa;between 50 kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDaand 500 kDa; between 100 kDa and 2,000 kDa; between 100 kDa and 1,750kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa;between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100kDa and 500 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa;between 200 kDa and 1,000 kDa; between 200 kDa and 750 kDa; or between200 kDa and 500 kDa. Any whole number integer within any of the aboveranges is contemplated as an embodiment of the disclosure.

In some embodiments, the serotype 33F glycoconjugate of the inventionhas a molecular weight of between 50 kDa and 20,000 kDa. In otherembodiments, the serotype 33F glycoconjugate has a molecular weight ofbetween 500 kDa and 10,000 kDa. In other embodiments, the serotype 33Fglycoconjugate has a molecular weight of between 200 kDa and 10,000 kDa.In still other embodiments, the serotype 33F glycoconjugate has amolecular weight of between 1,000 kDa and 3,000 kDa.

In further embodiments, the serotype 33F glycoconjugate of the inventionhas a molecular weight of between 200 kDa and 20,000 kDa; between 200kDa and 15,000 kDa; between 200 kDa and 10,000 kDa; between 200 kDa and7,500 kDa; between 200 kDa and 5,000 kDa; between 200 kDa and 3,000 kDa;between 200 kDa and 1,000 kDa; between 500 kDa and 20,000 kDa; between500 kDa and 15,000 kDa; between 500 kDa and 12,500 kDa; between 500 kDaand 10,000 kDa; between 500 kDa and 7,500 kDa; between 500 kDa and 6,000kDa; between 500 kDa and 5,000 kDa; between 500 kDa and 4,000 kDa;between 500 kDa and 3,000 kDa; between 500 kDa and 2,000 kDa; between500 kDa and 1,500 kDa; between 500 kDa and 1,000 kDa; between 750 kDaand 20,000 kDa; between 750 kDa and 15,000 kDa; between 750 kDa and12,500 kDa; between 750 kDa and 10,000 kDa; between 750 kDa and 7,500kDa; between 750 kDa and 6,000 kDa; between 750 kDa and 5,000 kDa;between 750 kDa and 4,000 kDa; between 750 kDa and 3,000 kDa; between750 kDa and 2,000 kDa; between 750 kDa and 1,500 kDa; between 1,000 kDaand 15,000 kDa; between 1,000 kDa and 12,500 kDa; between 1,000 kDa and10,000 kDa; between 1,000 kDa and 7,500 kDa; between 1,000 kDa and 6,000kDa; between 1,000 kDa and 5,000 kDa; between 1,000 kDa and 4,000 kDa;between 1,000 kDa and 2,500 kDa; between 2,000 kDa and 15,000 kDa;between 2,000 kDa and 12,500 kDa; between 2,000 kDa and 10,000 kDa;between 2,000 kDa and 7,500 kDa; between 2,000 kDa and 6,000 kDa;between 2,000 kDa and 5,000 kDa; between 2,000 kDa and 4,000 kDa;between 2,000 kDa and 3,000 kDa; between 3,000 kDa and 20,000 kDa;between 3,000 kDa and 15,000 kDa; between 3,000 kDa and 12,500 kDa;between 3,000 kDa and 10,000 kDa; between 3,000 kDa and 9,000 kDa;between 3,000 kDa and 8,000 kDa; between 3,000 kDa and 7,000 kDa;between 3,000 kDa and 6,000 kDa; between 3,000 kDa and 5,000 kDa orbetween 3,000 kDa and 4,000 kDa. Any whole number integer within any ofthe above ranges is contemplated as an embodiment of the disclosure.

Another way to characterize the serotype 33F glycoconjugates of theinvention is by the number of lysine residues in the carrier protein(e.g., CRM₁₉₇) that become conjugated to the saccharide, which can becharacterized as a range of conjugated lysines (degree of conjugation).

In a preferred embodiment, the degree of conjugation of the serotype 33Fglycoconjugate of the invention is between 2 and 20, between 4 and 16,between 2 and 15, between 2 and 13, between 2 and 10, between 2 and 8,between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 15,between 3 and 13, between 3 and 10, between 3 and 8, between 3 and 6,between 3 and 5, between 3 and 4, between 5 and 15, between 5 and 10,between 8 and 15, between 8 and 12, between 10 and 15 or between 10 and12. In an embodiment, the degree of conjugation of the serotype 33Fglycoconjugate of the invention is about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, about 15, about 16, about 17, about 18, about 19 or about20. In a preferred embodiment, the degree of conjugation of the serotype33F glycoconjugate of the invention is between 4 and 16. In some suchembodiments, the carrier protein is CRM₁₉₇.

In a preferred embodiment, the carrier protein comprises CRM₁₉₇, whichcontains 39 lysine residues. In some such embodiments, the CRM₁₉₇ maycomprise 4 to 16 lysine residues out of 39 covalently linked to thesaccharide. Another way to express this parameter is that about 10% toabout 41% of CRM₁₉₇ lysines are covalently linked to the saccharide. Inanother such embodiment, the CRM₁₉₇ may comprise 2 to 20 lysine residuesout of 39 covalently linked to the saccharide. Another way to expressthis parameter is that about 5% to about 50% of CRM₁₉₇ lysines arecovalently linked to the saccharide. In some embodiments, the CRM₁₉₇ maycomprise about 4, about 5, about 6, about 7, about 8, about 9, about 10,about 11, about 12, about 13, about 14, about 15, or about 16 lysineresidues out of 39 covalently linked to the saccharide.

In frequent embodiments, the carrier protein is covalently conjugated toan eTEC spacer through an amide linkage to one or more ε-amino groups oflysine residues on the carrier protein. In some such embodiments, thecarrier protein comprises 2 to 20 lysine residues covalently conjugatedto the saccharide. In other such embodiments, the carrier proteincomprises 4 to 16 lysine residues covalently conjugated to thesaccharide.

The serotype 33F glycoconjugates of the invention may also becharacterized by the ratio (weight/weight) of saccharide to carrierprotein. In some embodiments, the saccharide to carrier protein ratio(w/w) is between 0.2 and 4.0 (e.g., about 0.2, about 0.3, about 0.4,about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7,about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0,about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about3.7, about 3.8, about 3.9 or about 4.0). In other embodiments, thesaccharide to carrier protein ratio (w/w) is between 1.0 and 2.5. Infurther embodiments, the saccharide to carrier protein ratio (w/w) isbetween 0.4 and 1.7. In some such embodiments, the carrier protein isCRM₁₉₇.

The frequency of attachment of the saccharide chain to a lysine on thecarrier protein is another parameter for characterizing the serotype 33Fglycoconjugates of the invention. For example, in some embodiments, atleast one covalent linkage between the carrier protein and thepolysaccharide occurs for every 4 saccharide repeat units of thepolysaccharide. In another embodiment, the covalent linkage between thecarrier protein and the polysaccharide occurs at least once in every 10saccharide repeat units of the polysaccharide. In another embodiment,the covalent linkage between the carrier protein and the polysaccharideoccurs at least once in every 15 saccharide repeat units of thepolysaccharide. In a further embodiment, the covalent linkage betweenthe carrier protein and the polysaccharide occurs at least once in every25 saccharide repeat units of the polysaccharide.

In frequent embodiments, the carrier protein is CRM₁₉₇ and the covalentlinkage via an eTEC spacer between the CRM₁₉₇ and the polysaccharideoccurs at least once in every 4, 10, 15 or 25 saccharide repeat units ofthe polysaccharide.

In other embodiments, the conjugate comprises at least one covalentlinkage between the carrier protein and saccharide for every 5 to 10saccharide repeat units; every 2 to 7 saccharide repeat units; every 3to 8 saccharide repeat units; every 4 to 9 saccharide repeat units;every 6 to 11 saccharide repeat units; every 7 to 12 saccharide repeatunits; every 8 to 13 saccharide repeat units; every 9 to 14 sacchariderepeat units; every 10 to 15 saccharide repeat units; every 2 to 6saccharide repeat units, every 3 to 7 saccharide repeat units; every 4to 8 saccharide repeat units; every 6 to 10 saccharide repeat units;every 7 to 11 saccharide repeat units; every 8 to 12 saccharide repeatunits; every 9 to 13 saccharide repeat units; every 10 to 14 sacchariderepeat units; every 10 to 20 saccharide repeat units; every 4 to 25saccharide repeat units or every 2 to 25 saccharide repeat units. Infrequent embodiments, the carrier protein is CRM₁₉₇.

In another embodiment, at least one linkage between carrier protein andsaccharide occurs for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 saccharide repeat units ofthe polysaccharide. In an embodiment, the carrier protein is CRM₁₉₇. Anywhole number integer within any of the above ranges is contemplated asan embodiment of the disclosure.

An important consideration during conjugation is the development ofconditions that permit the retention of potentially sensitivenon-saccharide substituent functional groups of the individualcomponents, such as O-Acyl, phosphate or glycerol phosphate side chainsthat may form part of the saccharide epitope.

In one embodiment, the serotype 33F glycoconjugates of the inventioncomprise a saccharide which has a degree of O-acetylation between 10%and 100%. In some such embodiments, the saccharide has a degree ofO-acetylation between 50% and 100%. In other such embodiments, thesaccharide has a degree of O-acetylation between 75% and 100%. Infurther embodiments, the saccharide has a degree of 0-acetylationgreater than or equal to 70% (≥70%).

In a preferred embodiment, the serotype 33F glycoconjugate of theinvention comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 mMacetate per mM serotype 33F capsular polysaccharide. In a preferredembodiment, the glycoconjugate comprises at least 0.5, 0.6 or 0.7 mMacetate per mM serotype 33F capsular polysaccharide. In a preferredembodiment, the glycoconjugate comprises at least 0.6 mM acetate per mMserotype 33F capsular polysaccharide. In a preferred embodiment, theglycoconjugate comprises at least 0.7 mM acetate per mM serotype 33Fcapsular polysaccharide. In a preferred embodiment, the presence ofO-acetyl groups is determined by ion-HPLC analysis.

In a preferred embodiment, the ratio of mM acetate per mM serotype 33Fpolysaccharide in the glycoconjugate to mM acetate per mM serotype 33Fpolysaccharide in the isolated polysaccharide is at least 0.6, 0.65,0.7, 0.75, 0.8, 0.85, 0.9, or 0.95. In a preferred embodiment, the ratioof mM acetate per mM serotype 33F polysaccharide in the glycoconjugateto mM acetate per mM serotype 33F polysaccharide in the isolatedpolysaccharide is at least 0.7. In a preferred embodiment, the ratio ofmM acetate per mM serotype 33F polysaccharide in the glycoconjugate tomM acetate per mM serotype 33F polysaccharide in the isolatedpolysaccharide is at least 0.9.

In a preferred embodiment, the ratio of mM acetate per mM serotype 33Fpolysaccharide in the glycoconjugate to mM acetate per mM serotype 33Fpolysaccharide in the activated polysaccharide is at least 0.6, 0.65,0.7, 0.75, 0.8, 0.85, 0.9, or 0.95. In a preferred embodiment, the ratioof mM acetate per mM serotype 33F polysaccharide in the glycoconjugateto mM acetate per mM serotype 33F polysaccharide in the activatedpolysaccharide is at least 0.7. In a preferred embodiment, the ratio ofmM acetate per mM serotype 33F polysaccharide in the glycoconjugate tomM acetate per mM serotype 33F polysaccharide in the activatedpolysaccharide is at least 0.9.

The serotype 33F glycoconjugates and immunogenic compositions of theinvention may contain free saccharide that is not covalently conjugatedto the carrier protein, but is nevertheless present in theglycoconjugate composition. The free saccharide may be noncovalentlyassociated with (i.e., noncovalently bound to, adsorbed to, or entrappedin or with) the glycoconjugate.

In some embodiments, the serotype 33F glycoconjugates of the inventioncomprise less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or5% of free serotype 33F polysaccharide compared to the total amount ofserotype 33F polysaccharide. Preferably, the serotype 33F glycoconjugatecomprises less than 15% free saccharide, more preferably less than 10%free saccharide, and still more preferably, less than 5% of freesaccharide. In a preferred embodiment the serotype 33F glycoconjugatecomprises less than about 25% of free serotype 33F polysaccharidecompared to the total amount of serotype 33F polysaccharide. In apreferred embodiment the serotype 33F glycoconjugate comprises less thanabout 20% of free serotype 33F polysaccharide compared to the totalamount of serotype 33F polysaccharide. In a preferred embodiment theserotype 33F glycoconjugate comprises less than about 15% of freeserotype 33F polysaccharide compared to the total amount of serotype 33Fpolysaccharide.

In certain preferred embodiments, the invention provides a serotype 33Fglycoconjugate having one or more of the following features alone or incombination: the polysaccharide has a molecular weight of between 50 kDaand 2,000 kDa; the glycoconjugate has a molecular weight of between 500kDa to 10,000 KDa; the carrier protein comprises 2 to 20 lysine residuescovalently linked to the saccharide; the saccharide to carrier proteinratio (w/w) is between 0.2 and 4.0; the glycoconjugate comprises atleast one covalent linkage between the carrier protein and thepolysaccharide for every 4, 10, 15 or 25 saccharide repeat units of thepolysaccharide; the saccharide has a degree of O-acetylation between 75%and 100%; the conjugate comprises less than about 15% freepolysaccharide relative to total polysaccharide; the carrier protein isCRM₁₉₇.

The serotype 33F glycoconjugates may also be characterized by theirmolecular size distribution (K_(d)). Size exclusion chromatography media(CL-4B) can be used to determine the relative molecular sizedistribution of the conjugate, as mentioned above.

In an embodiment, at least 15% of the serotype 33F glycoconjugates ofthe invention have a K_(d) below or equal to 0.3 in a CL-4B column. Inan embodiment, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%,70%, 80% or 90% of the serotype 33F glycoconjugates of the inventionhave a K_(d) below or equal to 0.3 in a CL-4B column.

In a preferred embodiment, at least 35% of the serotype 33Fglycoconjugates of the invention have a K_(d) below or equal to 0.3 in aCL-4B column. In preferred embodiments, at least 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, or 85% of the serotype 33F glycoconjugates ofthe invention have a K_(d) below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 60% of the serotype 33F glycoconjugatesof the invention have a K_(d) below or equal to 0.3 in a CL-4B column.In a preferred embodiment, at least 70% of the serotype 33Fglycoconjugates of the invention have a K_(d) below or equal to 0.3 in aCL-4B column.

In a preferred embodiment, between 40% and 90% of the serotype 33Fglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column. Ina preferred embodiment, between 50% and 90% of the serotype 33Fglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column. Ina preferred embodiment, between 65% and 80% of the serotype 33Fglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column.

1.3.4 Glycoconjugates from S. pneumoniae Serotype 15B

In an embodiment, the serotype 15B glycoconjugates are obtained byactivating polysaccharide with 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate (CDAP) to form a cyanate ester. The activatedpolysaccharide may be coupled directly or via a spacer (linker) group toan amino group on the carrier protein. For example, the spacer could becystamine or cysteamine to give a thiolated polysaccharide which couldbe coupled to the carrier via a thioether linkage obtained afterreaction with a maleimide-activated carrier protein (for example usingGMBS) or a haloacetylated carrier protein (for example usingiodoacetimide, SIB, SIAB, sulfo-SIAB, SIA, or SBAP). Preferably, thecyanate ester (optionally made by CDAP chemistry) is coupled with hexanediamine or adipic acid dihydrazide (ADH) and the amino-derivatisedsaccharide is conjugated to the carrier protein using carbodiimide(e.g., EDAC or EDC) chemistry via a carboxyl group on the proteincarrier. Such conjugates are described for example in WO 93/15760, WO95/08348 and WO 96/129094.

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S--NHS, EDC, TSTU.Many are described in International Patent Application Publication No.WO 98/42721. Conjugation may involve a carbonyl linker which may beformed by reaction of a free hydroxyl group of the saccharide with CDI(see Bethell et al. (1979) J. Biol. Chem. 254:2572-2574; Hearn et al.(1981) J. Chromatogr. 218:509-518) followed by reaction with a proteinto form a carbamate linkage. This may involve reduction of the anomericterminus to a primary hydroxyl group, optional protection/deprotectionof the primary hydroxyl group, reaction of the primary hydroxyl groupwith CDI to form a CDI carbamate intermediate and coupling the CDIcarbamate intermediate with an amino group on a protein.

In preferred embodiments, the serotype 15B glycoconjugates of theinvention are prepared using reductive amination. Reductive aminationinvolves two steps: (1) oxidation of the polysaccharide to generatealdehyde functionalities from vicinal diols in individual hexasaccharideunit and (2) reduction of the activated polysaccharide and a carrierprotein to form a conjugate.

Preferably, before oxidation, sizing of the serotype 15B polysaccharideto a target molecular weight (MW) range is performed. Advantageously,the size of the purified serotype 15B polysaccharide is reduced whilepreserving critical features of the structure of the polysaccharide suchas for example the presence of O-acetyl groups. Preferably, the size ofthe purified serotype 15B polysaccharide is reduced by mechanicalhomogenization (see section 1.2.6 above).

The oxidation step may involve reaction with periodate. For the purposeof the present invention, the term “periodate” includes both periodateand periodic acid; the term also includes both metaperiodate (IO₄ ⁻) andorthoperiodate (IO₆ ⁵⁻) and the various salts of periodate (e.g., sodiumperiodate and potassium periodate). In a preferred embodiment theperiodate used for the oxidation of serotype 15B capsular polysaccharideis metaperiodate. In a preferred embodiment the periodate used for theoxidation of serotype 15B capsular polysaccharide is sodiummetaperiodate.

In a preferred embodiment, the polysaccharide is reacted with 0.01 to10.0, 0.05 to 5.0, 0.1 to 1.0, 0.5 to 1.0, 0.7 to 0.8, 0.05 to 0.5, 0.1to 0.3 molar equivalents of oxidizing agent. In a preferred embodiment,the polysaccharide is reacted with about 0.1, 0.15, 0.2, 0.25, 0.3,0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95molar equivalents of oxidizing agent. In a preferred embodiment, thepolysaccharide is reacted with about 0.15 molar equivalents of oxidizingagent. In a preferred embodiment, the polysaccharide is reacted withabout 0.25 molar equivalents of oxidizing agent. In a preferredembodiment, the polysaccharide is reacted with about 0.5 molarequivalents of oxidizing agent. In a preferred embodiment, thepolysaccharide is reacted with about 0.6 molar equivalents of oxidizingagent. In a preferred embodiment, the polysaccharide is reacted withabout 0.7 molar equivalents of oxidizing agent.

In a preferred embodiment, the duration of the reaction is between 1hour and 50 hours, between 10 hours and 30 hours, between 15 hours and20 hours, between 15 hours and 17 hours or about 16 hours.

In a preferred embodiment, the temperature of the reaction is maintainedbetween 15° C. and 45° C., between 15° C. and 30° C., between 20° C. and25° C. In a preferred embodiment, the temperature of the reaction ismaintained at about 23° C.

In a preferred embodiment, the oxidation reaction is carried out in abuffer selected from sodium phosphate, potassium phosphate,2-(N-morpholino)ethanesulfonic acid (MES) or Bis-Tris. In a preferredembodiment, the buffer is potassium phosphate.

In a preferred embodiment, the buffer has a concentration of between 1mM and 500 mM, between 1 mM and 300 mM, or between 50 mM and 200 mM. Ina preferred embodiment the buffer has a concentration of about 100 mM.

In a preferred embodiment, the oxidation reaction is carried out at a pHbetween 4.0 and 8.0, between 5.0 and 7.0, or between 5.5 and 6.5. In apreferred embodiment, the pH is about 6.0.

In preferred embodiment, the activated serotype 15B capsularpolysaccharide is obtained by reacting 0.5 mg/mL to 5 mg/mL of isolatedserotype 15B capsular polysaccharide with 0.2 to 0.3 molar equivalentsof periodate at a temperature between 20° C. and 25° C.

In a preferred embodiment, the activated serotype 15B capsularpolysaccharide is purified. The activated serotype 15B capsularpolysaccharide is purified according to methods known to the man skilledin the art, such as gel permeation chromatography (GPC), dialysis orultrafiltration/diafiltration. For example, the activated capsularpolysaccharide is purified by concentration and diafiltration using anultrafiltration device.

In a preferred embodiment, the degree of oxidation of the activatedserotype 15B capsular polysaccharide is between 2 and 20, between 2 and15, between 2 and 10, between 2 and 5, between 5 and 20, between 5 and15, between 5 and 10, between 10 and 20, between 10 and 15, or between15 and 20. In a preferred embodiment the degree of oxidation of theactivated serotype 15B capsular polysaccharide is between 2 and 10,between 4 and 8, between 4 and 6, between 6 and 8, between 6 and 12,between 8 and 12, between 9 and 11, between 10 and 16, between 12 and16, between 14 and 18, between 16 and 20, between 16 and 18, or between18 and 20.

In a preferred embodiment, the activated serotype 15B capsularpolysaccharide has a molecular weight between 5 kDa and 500 kDa, between50 kDa and 500 kDa, between 50 kDa and 450 kDa, between 100 kDa and 400kDa, between 100 kDa and 350 kDa. In a preferred embodiment, theactivated serotype 15B capsular polysaccharide has a molecular weightbetween 100 kDa and 350 kDa. In a preferred embodiment, the activatedserotype 15B capsular polysaccharide has a molecular weight between 100kDa and 300 kDa. In a preferred embodiment, the activated serotype 15Bcapsular polysaccharide has a molecular weight between 100 kDa and 250kDa.

In a preferred embodiment, the activated serotype 15B capsularpolysaccharide comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or0.8 mM acetate per mM of said serotype 15B capsular polysaccharide. In apreferred embodiment, the activated serotype 15B capsular polysaccharidecomprises at least 0.5, 0.6 or 0.7 mM acetate per mM of said serotype15B capsular polysaccharide. In a preferred embodiment, the activatedserotype 15B capsular polysaccharide comprises at least 0.6 mM acetateper mM of said serotype 15B capsular polysaccharide. In a preferredembodiment, the activated serotype 15B capsular polysaccharide comprisesat least 0.7 mM acetate per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the activated serotype 15B capsularpolysaccharide comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or0.8 mM glycerol per mM of said serotype 15B capsular polysaccharide. Ina preferred embodiment, the activated serotype 15B capsularpolysaccharide comprises at least 0.5, 0.6 or 0.7 mM glycerol per mM ofsaid serotype 15B capsular polysaccharide. In a preferred embodiment,the activated serotype 15B capsular polysaccharide comprises at least0.6 mM glycerol per mM of said serotype 15B capsular polysaccharide. Ina preferred embodiment, the activated serotype 15B capsularpolysaccharide comprises at least 0.7 mM glycerol per mM of saidserotype 15B capsular polysaccharide.

In a preferred embodiment, the activated serotype 15B capsularpolysaccharide has a molecular weight between 100 kDa and 250 kDa andcomprises at least 0.6 mM acetate per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the activated serotype 15B capsularpolysaccharide has a molecular weight between 100 kDa and 250 kDa andcomprises at least 0.6 mM glycerol per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the activated serotype 15B capsularpolysaccharide comprises at least 0.6 mM acetate per mM of said serotype15B capsular polysaccharide and at least 0.6 mM glycerol per mM of saidserotype 15B capsular polysaccharide.

In a preferred embodiment, the activated serotype 15B capsularpolysaccharide has a molecular weight between 100 kDa and 250 kDa andcomprises at least 0.6 mM acetate per mM of said serotype 15B capsularpolysaccharide and at least 0.6 mM glycerol per mM of said serotype 15Bcapsular polysaccharide.

In an embodiment, the activated serotype 15B capsular polysaccharide islyophilized, optionally in the presence of saccharide. In a preferredembodiment, the saccharide is selected from sucrose, trehalose,raffinose, stachyose, melezitose, dextran, mannitol, lactitol andpalatinit. In a preferred embodiment, the saccharide is sucrose. Thelyophilized activated capsular polysaccharide can then be compoundedwith a solution comprising the carrier protein.

In another embodiment, the activated serotype 15B capsularpolysaccharide is compounded with the carrier protein and lyophilizedoptionally in the presence of a saccharide. In a preferred embodiment,the saccharide is selected from sucrose, trehalose, raffinose,stachyose, melezitose, dextran, mannitol, lactitol and palatinit. In apreferred embodiment, the saccharide is sucrose. The co-lyophilizedpolysaccharide and carrier protein can then be resuspended in solutionand reacted with a reducing agent.

The activated serotype 15B capsular polysaccharide can be conjugated toa carrier protein by a process comprising the step of:

(a) compounding the activated serotype 15B capsular polysaccharide witha carrier protein, and(b) reacting the compounded activated serotype 15B capsularpolysaccharide and carrier protein with a reducing agent to form aserotype 15B capsular polysaccharide-carrier protein conjugate.

The conjugation of activated serotype 15B capsular polysaccharide with aprotein carrier by reductive amination in dimethylsulfoxide (DMSO) issuitable to preserve the O-acetyl content of the polysaccharide ascompared for example to reductive amination in aqueous solution wherethe level of O-acetylation of the polysaccharide is significantlyreduced. In a preferred embodiment, step (a) and step (b) are carriedout in DMSO.

In a preferred embodiment, step (a) comprises dissolving lyophilizedserotype 15B capsular polysaccharide in a solution comprising a carrierprotein and DMSO. In a preferred embodiment, step (a) comprisesdissolving co-lyophilized serotype 15B capsular polysaccharide andcarrier protein in DMSO.

When steps (a) and (b) are carried out in aqueous solution, steps (a)and (b) are carried out in a buffer, preferably selected from PBS, MES,HEPES, Bis-tris, ADA, PIPES, MOPSO, BES, MOPS, DIPSO, MOBS, HEPPSO,POPSO, TEA, EPPS, Bicine or HEPB, at a pH between 6.0 and 8.5, between7.0 and 8.0 or between 7.0 and 7.5. In a preferred embodiment the bufferis PBS. In a preferred embodiment the pH is about 7.3. In a preferredembodiment, the concentration of activated serotype 15B capsularpolysaccharide in step (b) is between 0.1 mg/mL and 10 mg/mL, between0.5 mg/mL and 5 mg/mL, or between 0.5 mg/mL and 2 mg/mL. In a preferredembodiment, the concentration of activated serotype 15B capsularpolysaccharide in step (b) is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0 mg/mL.

In a preferred embodiment the initial input ratio (weight by weight) ofactivated serotype 15B capsular polysaccharide to carrier protein isbetween 5:1 and 0.1:1, between 2:1 and 0.1:1, between 2:1 and 1:1,between 1.5:1 and 1:1, between 0.1:1 and 1:1, between 0.3:1 and 1:1, orbetween 0.6:1 and 1:1.

In a preferred embodiment the initial input ratio of activated serotype15B capsular polysaccharide to carrier protein is about 0.6:1 to 1:1. Inanother preferred embodiment the initial input ratio of activatedserotype 15B capsular polysaccharide to carrier protein is about 0.6:1to 1.5:1. Such initial input ratio is particularly suitable to obtainlow levels of free polysaccharide in the glycoconjugate.

In a preferred embodiment the initial input ratio of activated serotype15B capsular polysaccharide to carrier protein is about 0.4:1, 0.5:1,0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1,1.6:1, 1.7:1, 1.8:1, 1.9:1 or 2:1.

In an embodiment, the reducing agent is sodium cyanoborohydride, sodiumtriacetoxyborohydride, sodium or zinc borohydride in the presence ofBronsted or Lewis acids, amine boranes such as pyridine borane,2-Picoline Borane, 2,6-diborane-methanol, dimethylamine-borane,t-BuMeiPrN—BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane(PEMB). In a preferred embodiment, the reducing agent is sodiumcyanoborohydride. In a preferred embodiment, the reducing agent issodium 2-Picoline Borane.

In a preferred embodiment, the quantity of reducing agent used in step(b) is between about 0.1 and 10.0 molar equivalents, between 0.5 and 5.0molar equivalents, or between 1.0 and 2.0 molar equivalents. In apreferred embodiment, the quantity of reducing agent used in step (b) isabout 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 molarequivalents.

In a preferred embodiment, the duration of step (b) is between 1 hourand 60 hours, between 10 hours and 50 hours, between 40 hours and 50hours, or between 42 hours and 46 hours. In a preferred embodiment, theduration of step (b) is about 44 hours.

In a preferred embodiment, the temperature of the reaction in step (b)is maintained between 10° C. and 40° C., between 15° C. and 30° C. orbetween 20° C. and 26° C. In a preferred embodiment, the temperature ofthe reaction in step (b) is maintained at about 23° C.

In a preferred embodiment, the process for the preparation of aglycoconjugate comprising S. pneumoniae serotype 15B capsularpolysaccharide covalently linked to a carrier protein further comprisesa step (step (c)) of capping unreacted aldehyde (quenching) by additionof NaBH₄.

In a preferred embodiment, the quantity of NaBH₄ used in step (c) isbetween 0.1 and 10 molar equivalents, between 0.5 and 5.0 molarequivalents or between 1.0 and 3.0 molar equivalents. In a preferredembodiment, the quantity of NaBH₄ used in step (c) is about 2.0 molarequivalents.

In a preferred embodiment, the duration of step (c) is between 0.1 hoursand 10 hours, 0.5 hours and 5 hours, or between 2 hours and 4 hours. Ina preferred embodiment, the duration of step (c) is about 3 hours.

In a preferred embodiment, the temperature of the reaction in step (c)is maintained between 15° C. and 45° C., between 15° C. and 30° C. orbetween 20° C. and 26° C. In a preferred embodiment, the temperature ofthe reaction in step (c) is maintained at about 23° C.

In a preferred embodiment the yield of the conjugation step is greaterthan 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%. In a preferredembodiment the yield of the conjugation step (step b) is greater than60%. In a preferred embodiment the yield of the conjugation step (stepb) is greater than 70%. The yield is the amount of serotype 15Bpolysaccharide in the conjugate×100)/amount of activated polysaccharideused in the conjugation step.

In a preferred embodiment, the process for the preparation of aglycoconjugate comprising S. pneumoniae serotype 15B capsularpolysaccharide covalently linked to a carrier protein comprises thesteps of:

(a) sizing purified serotype 15B polysaccharide by high pressurehomogenization;(b) reacting the sized serotype 15B polysaccharide with an oxidizingagent;(c) compounding the activated serotype 15B polysaccharide with a carrierprotein;(d) reacting the compounded activated serotype 15B polysaccharide andcarrier protein with a reducing agent to form a serotype 15Bpolysaccharide-carrier protein conjugate; and(e) capping unreacted aldehyde (quenching) by addition of NaBH₄.

In a preferred embodiment the yield of the conjugation step (step d) ofthe above process is greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%or 90%. In a preferred embodiment the yield of the conjugation step(step d) is greater than 60%. In a preferred embodiment the yield of theconjugation step (step d) is greater than 70%. The yield is the amountof serotype 15B polysaccharide in the conjugate×100)/amount of activatedpolysaccharide used in the conjugation step.

After conjugation of the serotype 15B capsular polysaccharide to thecarrier protein, the polysaccharide-protein conjugate can be purified(enriched with respect to the amount of polysaccharide-proteinconjugate) by a variety of techniques known to the skilled person. Thesetechniques include dialysis, concentration/diafiltration operations,tangential flow filtration, precipitation/elution, column chromatography(DEAE or hydrophobic interaction chromatography), and depth filtration.

In an embodiment the carrier protein is as defined at section 1.1. In anembodiment the carrier protein is selected in the group consisting of:DT (Diphtheria toxin), TT (tetanus toxid), CRM₁₉₇, other DT mutants, PD(Haemophilus influenzae protein D), or immunologically functionalequivalents thereof. In an embodiment the carrier protein is CRM₁₉₇.

In some embodiments, the serotype 15B glycoconjugates of the presentinvention are conjugated to the carrier protein (e.g., CRM₁₉₇) andcomprise a saccharide having a molecular weight of between 5 kDa and1,500 kDa. In other such embodiments, the saccharide has a molecularweight of between 10 kDa and 1,500 kDa. In further such embodiments, thesaccharide has a molecular weight of between 50 kDa and 1,500 kDa;between 50 kDa and 1,250 kDa; between 50 kDa and 1,000 kDa; between 50kDa and 750 kDa; between 50 kDa and 500 kDa; between 50 kDa and 250 kDa;between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa; between100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100 kDa and500 kDa; between 100 kDa and 250 kDa; between 200 kDa and 1,500 kDa;between 200 kDa and 1,250 kDa; between 200 kDa and 1,000 kDa; between200 kDa and 750 kDa; or between 200 kDa and 500 kDa; or between 200 kDaand 400 kDa. Any whole number integer within any of the above ranges iscontemplated as an embodiment of the disclosure. In some embodiments,the serotype 15B glycoconjugate of the invention has a molecular weightof between 50 kDa and 20,000 kDa. In some embodiments, the serotype 15Bglycoconjugate of the invention has a molecular weight of between 1,000kDa and 20,000 kDa In a preferred embodiment, the serotype 15Bglycoconjugate of the invention has a molecular weight between 3,000 kDaand 20,000 kDa, between 5,000 kDa and 10,000 kDa, between 5,000 kDa and20,000 kDa, between 8,000 kDa and 20,000 kDa, between 8,000 kDa and16,000 kDa or between 10,000 kDa and 16,000 kDa.

In further embodiments, the serotype 15B glycoconjugate of the inventionhas a molecular weight of about 1,000 kDa, about 1,500 kDa, about 2,000kDa, about 2,500 kDa, about 3,000 kDa, about 3,500 kDa, about 4,000 kDa,about 4,500 kDa, about 5,000 kDa, about 5,500 kDa, about 6,000 kDa,about 6,500 kDa, about 7,000 kDa, about 7,500 kDa, about 8,000 kDa,about 8,500 kDa, about 9,000 kDa, about 9,500 kDa about 10,000 kDa,about 10,500 kDa, about 11,000 kDa, about 11,500 kDa, about 12,000 kDa,about 12,500 kDa, about 13,000 kDa, about 13,500 kDa, about 14,000 kDa,about 14,500 kDa, about 15,000 kDa, about 15,500 kDa, about 16,000 kDa,about 16,500 kDa, about 17,000 kDa, about 17,500 kDa, about 18,000 kDa,about 18,500 kDa about 19,000 kDa, about 19,500 kDa or about 20,000 kDa.

In further embodiments, the serotype 15B glycoconjugate of the inventionhas a molecular weight of between 1,000 kDa and 20,000 kDa; between1,000 kDa and 15,000 kDa; between 1,000 kDa and 10,000 kDa; between1,000 kDa and 7,500 kDa; between 1,000 kDa and 5,000 kDa; between 1,000kDa and 4,000 kDa; between 1,000 kDa and 3,000 kDa; between 2,000 kDaand 20,000 kDa; between 2,000 kDa and 15,000 kDa; between 2,000 kDa and12,500 kDa; between 2,000 kDa and 10,000 kDa; between 2,000 kDa and7,500 kDa; between 2,000 kDa and 6,000 kDa; between 2,000 kDa and 5,000kDa; between 2,000 kDa and 4,000 kDa; or between 2,000 kDa and 3,000kDa.

In further embodiments, the serotype 15B glycoconjugate of the inventionhas a molecular weight of between 3,000 kDa and 20,000 kDa; between3,000 kDa and 15,000 kDa; between 3,000 kDa and 10,000 kDa; between3,000 kDa and 7,500 kDa; between 3,000 kDa and 5,000 kDa; between 3,000kDa and 4,000 kDa; between 4,000 kDa and 20,000 kDa; between 4,000 kDaand 15,000 kDa; between 4,000 kDa and 12,500 kDa; between 4,000 kDa and10,000 kDa; between 4,000 kDa and 7,500 kDa; between 4,000 kDa and 6,000kDa or between 4,000 kDa and 5,000 kDa. In further embodiments, theserotype 15B glycoconjugate of the invention has a molecular weight ofbetween 5,000 kDa and 20,000 kDa; between 5,000 kDa and 15,000 kDa;between 5,000 kDa and 10,000 kDa; between 5,000 kDa and 7,500 kDa;between 6,000 kDa and 20,000 kDa; between 6,000 kDa and 15,000 kDa;between 6,000 kDa and 12,500 kDa; between 6,000 kDa and 10,000 kDa orbetween 6,000 kDa and 7,500 kDa.

The molecular weight of the glycoconjugate is measured by SEC-MALLS. Anywhole number integer within any of the above ranges is contemplated asan embodiment of the disclosure. In an embodiment, said serotype 15Bglycoconjugates are prepared using reductive amination.

The serotype 15B glycoconjugates of the invention may also becharacterized by the ratio (weight/weight) of saccharide to carrierprotein. In a preferred embodiment, the ratio (weight by weight) ofserotype 15B capsular polysaccharide to carrier protein in the conjugateis between 0.5 and 3.0 (e.g., about 0.5, about 0.6, about 0.7, about0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4,about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7,about 2.8, about 2.9 or about 3.0). In a preferred embodiment, the ratioof serotype 15B capsular polysaccharide to carrier protein in theconjugate is between 0.4 and 2. In a preferred embodiment, the ratio ofserotype 15B capsular polysaccharide to carrier protein in the conjugateis between 0.5 and 2.0, 0.5 and 1.5, 0.5 and 1.0, 1.0 and 1.5, 1.0 and2.0. In a preferred embodiment, the ratio of serotype 15B capsularpolysaccharide to carrier protein in the conjugate is between 0.7 and0.9. The serotype 15B glycoconjugates and immunogenic compositions ofthe invention may contain free saccharide that is not covalentlyconjugated to the carrier protein, but is nevertheless present in theglycoconjugate composition. The free saccharide may be noncovalentlyassociated with (i.e., noncovalently bound to, adsorbed to, or entrappedin or with) the glycoconjugate.

In a preferred embodiment, the serotype 15B glycoconjugate of theinvention comprises less than about 50%, 45%, 40%, 35%, 30%, 25%, 20% or15% of free serotype 15B capsular polysaccharide compared to the totalamount of serotype 15B capsular polysaccharide. In a preferredembodiment the serotype 15B glycoconjugate of the invention comprisesless than about 25% of free serotype 15B capsular polysaccharidecompared to the total amount of serotype 15B capsular polysaccharide. Ina preferred embodiment the serotype 15B glycoconjugate of the inventioncomprises less than about 20% of free serotype 15B capsularpolysaccharide compared to the total amount of serotype 15B capsularpolysaccharide. In a preferred embodiment the serotype 15Bglycoconjugates of the invention comprises less than about 15% of freeserotype 15B capsular polysaccharide compared to the total amount ofserotype 15B capsular polysaccharide.

The serotype 15B glycoconjugates may also be characterized by theirmolecular size distribution (K_(d)). Size exclusion chromatography media(CL-4B) can be used to determine the relative molecular sizedistribution of the conjugate, as mentioned above. In a preferredembodiment, at least 20% of the serotype 15B glycoconjugates of theinvention have a Kd below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 30% of the immunogenic conjugate has a Kdbelow or equal to 0.3 in a CL-4B column. In a preferred embodiment, atleast 40% of the serotype 15B glycoconjugates of the invention have aK_(d) below or equal to 0.3 in a CL-4B column. In a preferredembodiment, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% ofthe serotype 15 glycoconjugates of the invention have a K_(d) below orequal to 0.3 in a CL-4B column. In a preferred embodiment, at least 60%of the serotype 15B glycoconjugates of the invention have a K_(d) belowor equal to 0.3 in a CL-4B column. In a preferred embodiment, at least70% of the serotype 15B glycoconjugates of the invention have a K_(d)below or equal to 0.3 in a CL-4B column.

In a preferred embodiment, between 40% and 90% of the serotype 15Bglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column. Ina preferred embodiment, between 50% and 90% of the serotype 15Bglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column. Ina preferred embodiment, between 65% and 80% of the serotype 15Bglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column.

In a preferred embodiment, the serotype 15B glycoconjugate of theinvention comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 mMacetate per mM serotype 15B capsular polysaccharide. In a preferredembodiment, the glycoconjugate comprises at least 0.5, 0.6 or 0.7 mMacetate per mM serotype 15B capsular polysaccharide. In a preferredembodiment, the glycoconjugate comprises at least 0.6 mM acetate per mMserotype 15B capsular polysaccharide. In a preferred embodiment, theglycoconjugate comprises at least 0.7 mM acetate per mM serotype 15Bcapsular polysaccharide. In a preferred embodiment, the presence ofO-acetyl groups is determined by ion-HPLC analysis.

In a preferred embodiment, the ratio of mM acetate per mM serotype 15Bcapsular polysaccharide in the serotype 15B glycoconjugate to mM acetateper mM serotype 15B capsular polysaccharide in the isolatedpolysaccharide is at least 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or0.95. In a preferred embodiment, the ratio of mM acetate per mM serotype15B capsular polysaccharide in the serotype 15B glycoconjugate to mMacetate per mM serotype 15B capsular polysaccharide in the isolatedpolysaccharide is at least 0.7. In a preferred embodiment, the ratio ofmM acetate per mM serotype 15B capsular polysaccharide in the serotype15B glycoconjugate to mM acetate per mM serotype 15B capsularpolysaccharide in the isolated polysaccharide is at least 0.9. In apreferred embodiment, the presence of O-acetyl groups is determined byion-HPLC analysis.

In a preferred embodiment, the ratio of mM acetate per mM serotype 15Bcapsular polysaccharide in the serotype 15B glycoconjugate to mM acetateper mM serotype 15B capsular polysaccharide in the activatedpolysaccharide is at least 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or0.95. In a preferred embodiment, the ratio of mM acetate per mM serotype15B capsular polysaccharide in the serotype 15B glycoconjugate to mMacetate per mM serotype 15B capsular polysaccharide in the activatedpolysaccharide is at least 0.7. In a preferred embodiment, the ratio ofmM acetate per mM serotype 15B capsular polysaccharide in the serotype15B glycoconjugate to mM acetate per mM serotype 15B capsularpolysaccharide in the activated polysaccharide is at least 0.9. In apreferred embodiment, the presence of O-acetyl groups is determined byion-HPLC analysis.

In a preferred embodiment, the serotype 15B glycoconjugate of theinvention comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 mMglycerol per mM serotype 15B capsular polysaccharide. In a preferredembodiment, the serotype 15B glycoconjugate of the invention comprisesat least 0.5, 0.6 or 0.7 mM glycerol per mM serotype 15B capsularpolysaccharide. In a preferred embodiment, the serotype 15Bglycoconjugate of the invention comprises at least 0.6 mM glycerol permM serotype 15B capsular polysaccharide. In a preferred embodiment, theserotype 15B glycoconjugate of the invention comprises at least 0.7 mMglycerol per mM serotype 15B capsular polysaccharide.

Another way to characterize the serotype 15B glycoconjugates of theinvention is by the number of lysine residues in the carrier protein(e.g., CRM₁₉₇) that become conjugated to the saccharide which can becharacterized as a range of conjugated lysines (degree of conjugation).The evidence for lysine modification of the carrier protein, due tocovalent linkages to the polysaccharides, can be obtained by amino acidanalysis using routine methods known to those of skill in the art.Conjugation results in a reduction in the number of lysine residuesrecovered compared to the CRM₁₉₇ protein starting material used togenerate the conjugate materials.

In a preferred embodiment, the degree of conjugation of the serotype 15Bglycoconjugate of the invention is between 2 and 15, between 2 and 13,between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5,between 2 and 4, between 3 and 15, between 3 and 13, between 3 and 10,between 3 and 8, between 3 and 6, between 3 and 5, between 3 and 4,between 5 and 15, between 5 and 10, between 8 and 15, between 8 and 12,between 10 and 15 or between 10 and 12. In an embodiment, the degree ofconjugation of the serotype 15B glycoconjugate of the invention is about2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about10, about 11, about 12, about 13, about 14 or about 15. In a preferredembodiment, the degree of conjugation of the serotype 15B glycoconjugateof the invention is between 2 and 5.

1.3.5 Glycoconjugates from S. pneumoniae Serotype 12F

In the glycoconjugates from S. pneumoniae serotype 12F of the presentinvention, the saccharide is selected from the group consisting of apolysaccharide and an oligosaccharide, and the carrier protein isselected from any suitable carrier as described herein or known to thoseof skill in the art. In some preferred embodiments, the saccharide is apolysaccharide from S. pneumoniae serotype 12F.

In an embodiment, glycoconjugates from S. pneumoniae serotype 12F areprepared using CDAP. The polysaccharides are activated with1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form acyanate ester. The activated polysaccharide is then coupled directly orvia a spacer (linker) group to an amino group on the carrier protein(preferably CRM₁₉₇). For example, the spacer could be cystamine orcysteamine to give a thiolated polysaccharide which could be coupled tothe carrier via a thioether linkage obtained after reaction with amaleimide-activated carrier protein (for example using GMBS) or ahaloacetylated carrier protein (for example using iodoacetimide, SIB,SIAB, sulfo-SIAB, SIA, or SBAP). Preferably, the cyanate ester(optionally made by CDAP chemistry) is coupled with hexane diamine oradipic acid dihydrazide (ADH) and the amino-derivatised saccharide isconjugated to the carrier protein (e.g., CRM₁₉₇) using carbodiimide(e.g., EDAC or EDC) chemistry via a carboxyl group on the proteincarrier.

Other techniques for conjugation use carbodiimides, hydrazides, activeesters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S--NHS,EDC, TSTU. Many are described in International Patent ApplicationPublication No. WO 98/42721. Conjugation may involve a carbonyl linkerwhich may be formed by reaction of a free hydroxyl group of thesaccharide with CDI (see Bethell et al. (1979) J. Biol. Chem.254:2572-2574; Hearn et al. (1981) J. Chromatogr. 218:509-518) followedby reaction with a protein to form a carbamate linkage. This may involvereduction of the anomeric terminus to a primary hydroxyl group, optionalprotection/deprotection of the primary hydroxyl group, reaction of theprimary hydroxyl group with CDI to form a CDI carbamate intermediate andcoupling the CDI carbamate intermediate with an amino group on aprotein.

In an embodiment, capsular polysaccharides from serotypes 12F S.pneumoniae are conjugated to the carrier protein by reductive amination.Reductive amination involves two steps: (1) oxidation of thepolysaccharide to generate aldehyde functionalities from vicinal diolsin individual hexasaccharide unit and (2) reduction of the activatedpolysaccharide and a carrier protein to form a conjugate.

Before oxidation, the serotype 12F polysaccharide is optionallyhydrolized (sized). Mechanical or chemical hydrolysis maybe employed.Chemical hydrolysis maybe conducted using acetic acid.

In an embodiment, the oxidizing agent is periodate. The term “periodate”includes both periodate and periodic acid (see below).

In a preferred embodiment, the oxidizing agent is2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical andN-Chlorosuccinimide (NCS) as the cooxidant. In such embodiment, theglycoconjugates from S. pneumoniae serotype 12F are prepared using2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical to oxidizeprimary alcohols of the saccharide to aldehydes usingN-Chlorosuccinimide (NCS) as the cooxidant (hereinafter “TEMPO/NCSoxidation”), such as described at Example 7 and in WO 2014/097099.Therefore in one aspect, the glycoconjugates from S. pneumoniae serotype12F are obtainable by a method comprising the steps of: a) reacting a12F saccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) andN-chlorosuccinimide (NCS) in an aqueous solvent to produce an activatedsaccharide; and b) reacting the activated saccharide with a carrierprotein comprising one or more amine groups (hereinafter“TEMPO/NCS-reductive amination”). In one aspect, the glycoconjugatesfrom S. pneumoniae serotype 12F are obtained by said method. In anembodiment, the degree of oxidation of the activated 12F saccharideranges from 1 to 50, from 1 to 40, from 1 to 30, from 1 to 20, from 1 to10, from 1 to 5, from 3 to 40, from 3 to 30, from 3 to 20, from 3 to 10,from 4 to 40, from 4 to 30, from 4 to 20, from 4 to 10, from 5 to 30,from 5 to 25, from 5 to 20, from 5 to 10, from 6 to 50, from 6 to 40,from 6 to 30, from 6 to 20, from 6 to 15, from 6 to 14, from 6 to 13,from 6 to 12, from 6 to 11, from 6 to 10, from 7 to 40, from 7 to 30,from 7 to 20, from 7 to 15, from 7 to 14, from 7 to 13, from 7 to 12,from 7 to 11, from 7 to 10, from 8 to 40, from 8 to 30, from 8 to 20,from 8 to 15, from 8 to 14, from 8 to 13, from 8 to 12, from 8 to 11,from 8 to 10, from 9 to 40, from 9 to 30, from 9 to 20, from 9 to 15,from 10 to 40, from 10 to 30, from 10 to 20, or from 10 to 15. In afurther aspect, the degree of oxidation of the activated saccharide is1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, or 40. Preferably, the carrier protein is CRM₁₉₇.

In an embodiment, prior to step a), the 12F saccharide is hydrolyzed toa molecular weight ranging from 100 kDa to 400 kDa. For example, in oneaspect, the molecular weight ranges from 100 kDa to 350 kDa, from 100kDa to 300 kDa, from 100 kDa to 250 kDa, from 100 kDa to 200 kDa, from100 kDa to 150 kDa, from 200 kDa to 400 kDa, from 200 kDa to 350 kDa,from 200 kDa to 300 kDa, from 200 kDa to 250 kDa, from 300 kda to 400kDa, or from 300 kDa to 350 kDa.

In a further aspect, the method further comprises the step of purifyingthe activated polysaccharide prior to step b). In a further aspect, themethods further comprise the step of adding a reducing agent followingstep b). In one aspect, the reducing agent is NaCNBH₃. In a furtheraspect, the methods further comprise the step of adding NaBH₄ followingthe addition of NaCNBH₃. In a further aspect, the method comprises apurification step following the addition of NaBH₄.

In another aspect, the present disclosure provides a glycoconjugate fromS. pneumoniae serotype 12F produced, or obtainable by any of the methodsdisclosed hereabove. For example, in one aspect the present disclosureprovides a glycoconguate from S. pneumoniae serotype 12F comprising asaccharide conjugated to a carrier protein that is produced orobtainable by the method comprising the steps of: a) reacting asaccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) andN-chlorosuccinimide (NCS) in an aqueous solvent to produce an activatedsaccharide; and b) reacting the activated saccharide with a carrierprotein comprising one or more amine groups.

In one embodiment, the glycoconjugate from S. pneumoniae serotype 12F ofthe present invention has a molecular weight of between about 50 kDa andabout 20,000 kDa. In another embodiment, the glycoconjugate has amolecular weight of between about 200 kDa and about 10,000 kDa. Inanother embodiment, the glycoconjugate from S. pneumoniae serotype 12Fhas a molecular weight of between about 500 kDa and about 5,000 kDa. Inone embodiment, the glycoconjugate from S. pneumoniae serotype 12F has amolecular weight of between about 1,000 kDa and about 3,000 kDa. Inother embodiments the glycoconjugate from S. pneumoniae serotype 12F hasa molecular weight of between about 600 kDa and about 2,800 kDa; betweenabout 700 kDa and about 2,700 kDa; between about 1,000 kDa and about2,000 kDa; between about 1,800 kDa and about 2,500 kDa; between about1,100 kDa and about 2,200 kDa; between about 1,900 kDa and about 2,700kDa; between about 1,200 kDa and about 2,400 kDa; between about 1,700kDa and about 2,600 kDa; between about 1,300 kDa and about 2,600 kDa;between about 1,600 kDa and about 3,000 kDa.

In further embodiments, the serotype 12F glycoconjugate of the inventionhas a molecular weight of between 1,000 kDa and 20,000 kDa; between1,000 kDa and 15,000 kDa; between 1,000 kDa and 10,000 kDa; between1,000 kDa and 7,500 kDa; between 1,000 kDa and 5,000 kDa; between 1,000kDa and 4,000 kDa; between 1,000 kDa and 3,000 kDa; between 2,000 kDaand 20,000 kDa; between 2,000 kDa and 15,000 kDa; between 2,000 kDa and12,500 kDa; between 2,000 kDa and 10,000 kDa; between 2,000 kDa and7,500 kDa; between 2,000 kDa and 6,000 kDa; between 2,000 kDa and 5,000kDa; between 2,000 kDa and 4,000 kDa; or between 2,000 kDa and 3,000kDa. Any whole number integer within any of the above ranges iscontemplated as an embodiment of the disclosure. In some suchembodiments, the carrier protein is CRM₁₉₇. In some such embodiments,the serotype 12F glycoconjugate is conjugated to the carrier protein byTEMPO/NCS-reductive amination.

Another way to characterize the serotype 12F glycoconjugates of theinvention is by the number of lysine residues in the carrier protein(e.g., CRM₁₉₇) that become conjugated to the saccharide, which can becharacterized as a range of conjugated lysines (degree of conjugation).

In a preferred embodiment, the degree of conjugation of the serotype 12Fglycoconjugate of the invention is between 2 and 20, between 4 and 16,between 4 and 15, between 2 and 15, between 2 and 13, between 2 and 10,between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4,between 3 and 15, between 3 and 13, between 3 and 10, between 3 and 8,between 3 and 6, between 3 and 5, between 3 and 4, between 5 and 15,between 5 and 10, between 8 and 15, between 8 and 12, between 10 and 15or between 10 and 12. In an embodiment, the degree of conjugation of theserotype 12F glycoconjugate of the invention is about 2, about 3, about4, about 5, about 6, about 7, about 8, about 9, about 10, about 11,about 12, about 13, about 14, about 15, about 16, about 17, about 18,about 19 or about 20.

The number of lysine residues in the carrier protein conjugated to thesaccharide may also be expressed as a molar ratio. For example, in aglycoconjugate where 4 to 15 lysine residues of CRM₁₉₇ are covalentlylinked to the saccharide, the molar ratio of conjugated lysines toCRM₁₉₇ in the glycoconjugate is between about 10:1 to about 40:1. In animmunogenic composition where 2 to 20 lysine residues of CRM₁₉₇ arecovalently linked to the saccharide, the molar ratio of conjugatedlysines to CRM₁₉₇ in the glycoconjugate is between about 5:1 and about50:1. In one embodiment, in the glycoconjugate from S. pneumoniaeserotype 12F of the present invention the molar ratio of conjugatedlysines to carrier protein is from about 10:1 to about 25:1. In somesuch embodiments, the carrier protein is CRM₁₉₇. In some embodiments,the CRM₁₉₇ may comprise about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,or 16 lysine residues out of 39 covalently linked to the saccharide. Insome such embodiments, the serotype 12F glycoconjugate is conjugated tothe carrier protein by TEMPO/NCS-reductive amination.

In one embodiment, the saccharide to carrier protein ratio (w/w) isbetween 0.2 and 4.0 in the glycoconjugate from S. pneumoniae serotype12F (e.g., about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3,about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6,about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9 orabout 4.0). In another embodiment, the saccharide to carrier proteinratio (w/w) is between 1.1 and 1.7 in the glycoconjugate from S.pneumoniae serotype 12F. In other embodiments, the saccharide to carrierprotein ratio (w/w) is between 0.8 and 1.8 (e.g., about 0.8, about 0.9,about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about1.6, about 1.7 or about 1.8). In some such embodiments, the carrierprotein is CRM₁₉₇. In some such embodiments, the carrier protein isCRM₁₉₇. In some such embodiments, the serotype 12F glycoconjugate isconjugated to the carrier protein by TEMPO/NCS-reductive amination.

The frequency of attachment of the saccharide chain to a lysine on thecarrier protein is another parameter for characterizing the serotype 12Fglycoconjugates of the disclosure. For example, in one embodiment, thereis at least one covalent linkage between the carrier protein and thepolysaccharide for every 100 saccharide repeat units of thepolysaccharide. In one embodiment, there is at least one covalentlinkage between the carrier protein and the polysaccharide for every 50saccharide repeat units of the polysaccharide. In one embodiment, thereis at least one covalent linkage between the carrier protein and thepolysaccharide for every 25 saccharide repeat units of thepolysaccharide. In another embodiment, the covalent linkage between thecarrier protein and the polysaccharide occurs at least once in every 4saccharide repeat units of the polysaccharide. In another embodiment,the covalent linkage between the carrier protein and the polysaccharideoccurs at least once in every 10 saccharide repeat units of thepolysaccharide. In a further embodiment, the covalent linkage betweenthe carrier protein and the polysaccharide occurs at least once in every15 saccharide repeat units of the polysaccharide. In frequentembodiments, the carrier protein is CRM₁₉₇ and the covalent linkagebetween the CRM₁₉₇ and the polysaccharide occurs at least once in every4, 10, 15 or 25 saccharide repeat units of the polysaccharide.

In other embodiments, the conjugate comprises at least one covalentlinkage between the carrier protein and saccharide for every 5 to 10saccharide repeat units; every 2 to 7 saccharide repeat units; every 3to 8 saccharide repeat units; every 4 to 9 saccharide repeat units;every 6 to 11 saccharide repeat units; every 7 to 12 saccharide repeatunits; every 8 to 13 saccharide repeat units; every 9 to 14 sacchariderepeat units; every 10 to 15 saccharide repeat units; every 2 to 6saccharide repeat units, every 3 to 7 saccharide repeat units; every 4to 8 saccharide repeat units; every 6 to 10 saccharide repeat units;every 7 to 11 saccharide repeat units; every 8 to 12 saccharide repeatunits; every 9 to 13 saccharide repeat units; every 10 to 14 sacchariderepeat units; every 10 to 20 saccharide repeat units; every 4 to 25saccharide repeat units or every 2 to 25 saccharide repeat units. Infrequent embodiments, the carrier protein is CRM₁₉₇.

In another embodiment, at least one linkage between CRM₁₉₇ andsaccharide occurs for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 saccharide repeat units ofthe polysaccharide. In some such embodiments, the serotype 12Fglycoconjugate is conjugated to the carrier protein byTEMPO/NCS-reductive amination.

In one embodiment, the glycoconjugate from S. pneumoniae serotype 12F ofthe invention comprises at least one covalent linkage between thecarrier protein and the polysaccharide for every 25 saccharide repeatunits of the polysaccharide. In another embodiment, the covalent linkagebetween the carrier protein and the polysaccharide occurs at least oncein every 4 saccharide repeat units of the polysaccharide. In anotherembodiment, the covalent linkage between the carrier protein and thepolysaccharide occurs at least once in every 10 saccharide repeat unitsof the polysaccharide. In a further embodiment, the covalent linkagebetween the carrier protein and the polysaccharide occurs at least oncein every 15 saccharide repeat units of the polysaccharide. In some suchembodiments, the serotype 12F glycoconjugate is conjugated to thecarrier protein by TEMPO/NCS-reductive amination.

The serotype 12F glycoconjugates and immunogenic compositions of theinvention may contain free saccharide that is not covalently conjugatedto the carrier protein, but is nevertheless present in theglycoconjugate composition. The free saccharide may be noncovalentlyassociated with (i.e., noncovalently bound to, adsorbed to, or entrappedin or with) the glycoconjugate.

In some embodiments, the serotype 12F glycoconjugates of the inventioncomprise less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or5% of free serotype 12F polysaccharide compared to the total amount ofserotype 12F polysaccharide. In one embodiment, the glycoconjugate fromS. pneumoniae serotype 12F comprises less than about 50% of freeserotype 12F polysaccharide compared to the total amount of serotype 12Fpolysaccharide. In one embodiment, the glycoconjugate from S. pneumoniaeserotype 12F comprises less than about 45% of free serotype 12Fpolysaccharide compared to the total amount of serotype 12Fpolysaccharide. In another embodiment, the glycoconjugate comprises lessthan about 30% of free serotype 12F polysaccharide compared to the totalamount of serotype 12F polysaccharide. In another embodiment, theglycoconjugate from S. pneumoniae serotype 12F comprises less than about20% of free serotype 12F polysaccharide compared to the total amount ofserotype 12F polysaccharide. In a further embodiment, the glycoconjugatecomprises less than about 10% of free serotype 12F polysaccharidecompared to the total amount of serotype 12F polysaccharide. In anotherembodiment, the glycoconjugate from S. pneumoniae serotype 12F comprisesless than about 5% of free serotype 12F polysaccharide compared to thetotal amount of serotype 12F polysaccharide. In some such embodiments,the serotype 12F glycoconjugate is conjugated to the carrier protein byTEMPO/NCS-reductive amination.

In some embodiments, the serotype 12F glycoconjugate of the presentinvention comprises a saccharide having a molecular weight of between 10kDa and 2,000 kDa. In other such embodiments, the saccharide has amolecular weight of between 50 kDa and 2,000 kDa. In further suchembodiments, the saccharide has a molecular weight of between 50 kDa and1,750 kDa; between 50 kDa and 1,500 kDa; between 50 kDa and 1,250 kDa;between 50 kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDaand 500 kDa; between 100 kDa and 2,000 kDa; between 100 kDa and 1,750kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa;between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100kDa and 500 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa;between 200 kDa and 1,000 kDa; between 200 kDa and 750 kDa; or between200 kDa and 500 kDa; or between 200 kDa and 400 kDa. In some suchembodiments, the serotype 12F glycoconjugate is conjugated to thecarrier protein by TEMPO/NCS-reductive amination.

The serotype 12F glycoconjugates may also be characterized by theirmolecular size distribution (K_(d)). Size exclusion chromatography media(CL-4B) can be used to determine the relative molecular sizedistribution of the conjugate, as mentioned above. In a preferredembodiment, at least 35% of the serotype 12F glycoconjugates of theinvention have a K_(d) below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, or 85% of the serotype 12F glycoconjugates of the invention have aK_(d) below or equal to 0.3 in a CL-4B column. In a preferredembodiment, at least 60% of the serotype 12F glycoconjugates of theinvention have a K_(d) below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 70% of the serotype 12F glycoconjugatesof the invention have a K_(d) below or equal to 0.3 in a CL-4B column.

In a preferred embodiment, between 40% and 90% of the serotype 12Fglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column. Ina preferred embodiment, between 50% and 90% of the serotype 12Fglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column. Ina preferred embodiment, between 65% and 80% of the serotype 12Fglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column.

1.3.6 Glycoconjugates from S. pneumoniae Serotype 10A

In an embodiment, the serotype 10A glycoconjugates are obtained byactivating polysaccharide with 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate (CDAP) to form a cyanate ester. The activatedpolysaccharide may be coupled directly or via a spacer (linker) group toan amino group on the carrier protein. For example, the spacer could becystamine or cysteamine to give a thiolated polysaccharide which couldbe coupled to the carrier via a thioether linkage obtained afterreaction with a maleimide-activated carrier protein (for example usingGMBS) or a haloacetylated carrier protein (for example usingiodoacetimide, SIB, SIAB, sulfo-SIAB, SIA, or SBAP). Preferably, thecyanate ester (optionally made by CDAP chemistry) is coupled with hexanediamine or adipic acid dihydrazide (ADH) and the amino-derivatisedsaccharide is conjugated to the carrier protein using carbodiimide(e.g., EDAC or EDC) chemistry via a carboxyl group on the proteincarrier. Such conjugates are described for example in WO 93/15760, WO95/08348 and WO 96/129094.

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S--NHS, EDC, TSTU.Many are described in International Patent Application Publication No.WO 98/42721. Conjugation may involve a carbonyl linker which may beformed by reaction of a free hydroxyl group of the saccharide with CDI(See Bethell et al. (1979) J. Biol. Chem. 254:2572-2574; Hearn et al.(1981) J. Chromatogr. 218:509-518) followed by reaction with a proteinto form a carbamate linkage. This may involve reduction of the anomericterminus to a primary hydroxyl group, optional protection/deprotectionof the primary hydroxyl group, reaction of the primary hydroxyl groupwith CDI to form a CDI carbamate intermediate and coupling the CDIcarbamate intermediate with an amino group on a protein.

In preferred embodiments, the serotype 10A glycoconjugates of theinvention are prepared using reductive amination. Reductive aminationinvolves two steps: (1) oxidation of the polysaccharide to generatealdehyde functionalities from vicinal diols in individual hexasaccharideunit and (2) reduction of the activated polysaccharide and a carrierprotein to form a conjugate.

Before oxidation, the serotype 10A polysaccharide is optionallyhydrolized (sized). Mechanical or chemical hydrolysis maybe employed.Chemical hydrolysis maybe conducted using acetic acid.

In an embodiment, serotype polysaccharide is activated (oxidized) by aprocess comprising the step of:

(a) reacting isolated serotype 10A polysaccharide with an oxidizingagent; and(b) quenching the oxidation reaction by addition of a quenching agentresulting in an activated serotype 10A polysaccharide.

In a preferred embodiment, the oxidizing agent is periodate. For thepurpose of the present invention, the term “periodate” includes bothperiodate and periodic acid, the term also includes both metaperiodate(IO₄ ⁻) and orthoperiodate (IO₆ ⁵⁻) and the various salts of periodate(e.g., sodium periodate and potassium periodate). In a preferredembodiment, the oxidizing agent is sodium periodate. In a preferredembodiment, the periodate used for the oxidation of serotype 10Apolysaccharide is metaperiodate. In a preferred embodiment the periodateused for the oxidation of serotype 10A polysaccharide is sodiummetaperiodate.

In one embodiment, the quenching agent is selected from vicinal diols,1,2-aminoalcohols, amino acids, glutathione, sulfite, bisulfate,dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites orphosphorous acid.

In one embodiment, the quenching agent is a 1,2-aminoalcohols of formula(I):

wherein R¹ is selected from H, methyl, ethyl, propyl or isopropyl.

In one embodiment, the quenching agent is selected from sodium andpotassium salts of sulfite, bisulfate, dithionite, metabisulfite,thiosulfate, phosphites, hypophosphites or phosphorous acid.

In one embodiment, the quenching agent is an amino acid. In suchembodiments, said amino acid may be selected from serine, threonine,cysteine, cystine, methionine, proline, hydroxyproline, tryptophan,tyrosine, and histidine.

In one embodiment, the quenching agent is a sulfite such as bisulfate,dithionite, metabisulfite, thiosulfate.

In one embodiment, the quenching agent is a compound comprising twovicinal hydroxyl groups (vicinal diols), i.e., two hydroxyl groupscovalently linked to two adjacent carbon atoms.

Preferably, the quenching agent is a compound of formula (II):

wherein R¹ and R² are each independently selected from H, methyl, ethyl,propyl or isopropyl.

In a preferred embodiment, the quenching agent is glycerol, ethyleneglycol, propan-1,2-diol, butan-1,2-diol or butan-2,3-diol, ascorbicacid. In a preferred embodiment, the quenching agent is butan-2,3-diol.

In preferred embodiment, the isolated serotype 10A polysaccharide isactivated by a process comprising the step of:

(a) reacting isolated serotype 10A polysaccharide with periodate; and(b) quenching the oxidation reaction by addition of butan-2,3-diolresulting in an activated serotype 10A polysaccharide.

Following the oxidation step of the polysaccharide, the polysaccharideis said to be activated and is referred to an “activated polysaccharide”hereinafter.

In a preferred embodiment, the activated serotype 10A polysaccharide ispurified. The activated serotype 10A polysaccharide is purifiedaccording to methods known to the man skilled in the art, such as gelpermeation chromatography (GPC), dialysis orultrafiltration/diafiltration. For example, the activated 10Apolysaccharide is purified by concentration and diafiltration using anultrafiltration device.

In a preferred embodiment the degree of oxidation of the activatedserotype 10A polysaccharide is between 2 and 30, between 2 and 25,between 2 and 20, between 2 and 15, between 2 and 10, between 2 and 5,between 5 and 30, between 5 and 25, between 5 and 20, between 5 and 15,between 5 and 10, between 10 and 30, between 10 and 25, between 10 and20, between 10 and 15, between 15 and 30, between 15 and 25, between 15and 20, between 20 to 30, or between 20 to 25. In a preferred embodimentthe degree of oxidation of the activated serotype 10A polysaccharide isbetween 2 and 10, between 4 and 8, between 4 and 8, between 4 and 6,between 6 and 8, between 6 and 12, between 8 and 14, between 9 and 11,between 10 and 16, between 12 and 16, between 14 and 18, between 16 and20, between 16 and 18, between 18 and 22, or between 18 and 20.

In a preferred embodiment, the activated serotype 10A polysaccharide hasa molecular weight between 50 kDa and 400 kDa, between 50 kDa and 350kDa, between 50 kDa and 300 kDa, between 50 kDa and 250 kDa, between 50kDa and 200 kDa, between 100 kDa and 300 kDa, between 100 kDa and 250kDa or between 100 kDa and 200 kDa. In a preferred embodiment, theactivated serotype 10A polysaccharide has a molecular weight between 50kDa and 300 kDa. In a preferred embodiment, the activated serotype 10Apolysaccharide has a molecular weight between 100 kDa and 200 kDa. In apreferred embodiment, the activated serotype 10A polysaccharide has amolecular weight between 100 kDa and 200 kDa and a degree of oxidationbetween 5 and 20, between 5 and 15, between 8 and 14, between 8 and 12or between 9 and 11. In a preferred embodiment, the activated serotype10A polysaccharide has a molecular weight between 100 kDa and 200 kDaand a degree of oxidation between 9 and 11.

The activated polysaccharide and/or the carrier protein may belyophilised (freeze-dried), either independently (discretelyophilization) or together (co-lyophilized).

In an embodiment, the activated serotype 10A polysaccharide islyophilized, optionally in the presence of saccharide. In a preferredembodiment, the saccharide is selected from sucrose, trehalose,raffinose, stachyose, melezitose, dextran, mannitol, lactitol andpalatinit. In a preferred embodiment, the saccharide is sucrose. In oneembodiment, the lyophilized activated polysaccharide is then compoundedwith a solution comprising the carrier protein.

In another embodiment the activated polysaccharide and the carrierprotein are co-lyophilised. In such embodiments, the activated serotype10A polysaccharide is compounded with the carrier protein andlyophilized optionally in the presence of a saccharide. In a preferredembodiment, the saccharide is selected from sucrose, trehalose,raffinose, stachyose, melezitose, dextran, mannitol, lactitol andpalatinit. In a preferred embodiment, the saccharide is sucrose. Theco-lyophilized polysaccharide and carrier protein can then beresuspended in solution and reacted with a reducing agent.

The second step of the conjugation process is the reduction of theactivated polysaccharide and a carrier protein to form a conjugate(reductive amination), using a reducing agent.

The activated serotype 10A polysaccharide can be conjugated to a carrierprotein by a process comprising the step of:

(c) compounding the activated serotype 10A polysaccharide with a carrierprotein; and(d) reacting the compounded activated serotype 10A polysaccharide andcarrier protein with a reducing agent to form a serotype 10Apolysaccharide-carrier protein conjugate.

In an embodiment, the reduction reaction is carried out in aqueoussolvent, in another embodiment the reaction is carried out in aproticsolvent. In an embodiment, the reduction reaction is carried out in DMSO(dimethylsulfoxide) or in DMF (dimethylformamide) solvent. The DMSO orDMF solvent may be used to reconstitute the activated polysaccharide andcarrier protein which has been lyophilised.

In an embodiment, the reducing agent is sodium cyanoborohydride, sodiumtriacetoxyborohydride, sodium or zinc borohydride in the presence ofBronsted or Lewis acids, amine boranes such as pyridine borane,2-Picoline Borane, 2,6-diborane-methanol, dimethylamine-borane,t-BuMeiPrN—BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane(PEMB). In a preferred embodiment, the reducing agent is sodiumcyanoborohydride.

At the end of the reduction reaction, there may be unreacted aldehydegroups remaining in the conjugates, these may be capped using a suitablecapping agent. In one embodiment this capping agent is sodiumborohydride (NaBH₄).

Following conjugation of serotype 10A polysaccharide to the carrierprotein, the glycoconjugate can be purified (enriched with respect tothe amount of polysaccharide-protein conjugate) by a variety oftechniques known to the skilled person. These techniques includedialysis, concentration/diafiltration operations, tangential flowfiltration precipitation/elution, column chromatography (DEAE orhydrophobic interaction chromatography), and depth filtration.

In some embodiments, the serotype 10A glycoconjugates of the presentinvention comprise a saccharide having a molecular weight of between 10kDa and 2,000 kDa. In other such embodiments, the saccharide has amolecular weight of between 50 kDa and 2,000 kDa. In further suchembodiments, the saccharide has a molecular weight of between 50 kDa and1,750 kDa; between 50 kDa and 1,500 kDa; between 50 kDa and 1,250 kDa;between 50 kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDaand 500 kDa; between 100 kDa and 2,000 kDa; between 100 kDa and 1,750kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa;between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100kDa and 500 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa;between 200 kDa and 1,000 kDa; between 200 kDa and 750 kDa; or between200 kDa and 500 kDa; or between 200 kDa and 400 kDa. In some suchembodiments, the serotype 10A glycoconjugates are prepared usingreductive amination.

In some embodiments, the serotype 10A glycoconjugate of the inventionhas a molecular weight of between 50 kDa and 20,000 kDa. In otherembodiments, the serotype 10A glycoconjugate has a molecular weight ofbetween 50 kDa and 15,000 kDa. In other embodiments, the serotype 10Aglycoconjugate has a molecular weight of between 500 kDa and 15,000 kDa,between 500 kDa and 10,000 kDa; between 2,000 kDa and 10,000 kDa; orbetween 3,000 kDa and 8,000 kDa. In other embodiments, the serotype 10Aglycoconjugate has a molecular weight of between 1,000 kDa and 10,000kDa. In other embodiments, the serotype 10A glycoconjugate has amolecular weight of between 1,000 kDa and 8,000 kDa. In still otherembodiments, the the serotype 10A glycoconjugate has a molecular weightof between 2,000 kDa and 8,000 kDa or between 3,000 kDa and 7,000 kDa.In further embodiments, the serotype 10A glycoconjugate of the inventionhas a molecular weight of between 200 kDa and 20,000 kDa; between 200kDa and 15,000 kDa; between 200 kDa and 10,000 kDa; between 200 kDa and7,500 kDa; between 200 kDa and 5,000 kDa; between 200 kDa and 3,000 kDa;between 200 kDa and 1,000 kDa; between 500 kDa and 20,000 kDa; between500 kDa and 15,000 kDa; between 500 kDa and 12,500 kDa; between 500 kDaand 10,000 kDa; between 500 kDa and 7,500 kDa; between 500 kDa and 6,000kDa; between 500 kDa and 5,000 kDa; between 500 kDa and 4,000 kDa;between 500 kDa and 3,000 kDa; between 500 kDa and 2,000 kDa; between500 kDa and 1,500 kDa; between 500 kDa and 1,000 kDa; between 750 kDaand 20,000 kDa; between 750 kDa and 15,000 kDa; between 750 kDa and12,500 kDa; between 750 kDa and 10,000 kDa; between 750 kDa and 7,500kDa; between 750 kDa and 6,000 kDa; between 750 kDa and 5,000 kDa;between 750 kDa and 4,000 kDa; between 750 kDa and 3,000 kDa; between750 kDa and 2,000 kDa; between 750 kDa and 1,500 kDa; between 1,000 kDaand 15,000 kDa; between 1,000 kDa and 12,500 kDa; between 1,000 kDa and10,000 kDa; between 1,000 kDa and 7,500 kDa; between 1,000 kDa and 6,000kDa; between 1,000 kDa and 5,000 kDa; between 1,000 kDa and 4,000 kDa;between 1,000 kDa and 2,500 kDa; between 2,000 kDa and 15,000 kDa;between 2,000 kDa and 12,500 kDa; between 2,000 kDa and 10,000 kDa;between 2,000 kDa and 7,500 kDa; between 2,000 kDa and 6,000 kDa;between 2,000 kDa and 5,000 kDa; between 2,000 kDa and 4,000 kDa; orbetween 2,000 kDa and 3,000 kDa.

In further embodiments, the serotype 10A glycoconjugate of the inventionhas a molecular weight of between 3,000 kDa and 20,000 kDa; between3,000 kDa and 15,000 kDa; between 3,000 kDa and 10,000 kDa; between3,000 kDa and 7,500 kDa; between 3,000 kDa and 5,000 kDa; between 4,000kDa and 20,000 kDa; between 4,000 kDa and 15,000 kDa; between 4,000 kDaand 12,500 kDa; between 4,000 kDa and 10,000 kDa; between 4,000 kDa and7,500 kDa; between 4,000 kDa and 6,000 kDa; or between 4,000 kDa and5,000 kDa. In further embodiments, the serotype 10A glycoconjugate ofthe invention has a molecular weight of between 5,000 kDa and 20,000kDa; between 5,000 kDa and 15,000 kDa; between 5,000 kDa and 10,000 kDaor between 5,000 kDa and 7,500 kDa. In further embodiments, the serotype10A glycoconjugate of the invention has a molecular weight of between6,000 kDa and 20,000 kDa; between 6,000 kDa and 15,000 kDa; between6,000 kDa and 10,000 kDa or between 6,000 kDa and 7,500 kDa. In furtherembodiments, the serotype 10A glycoconjugate of the invention has amolecular weight of between 7,000 kDa and 20,000 kDa; between 7,000 kDaand 15,000 kDa; between 7,000 kDa and 10,000 kDa or between 7,000 kDaand 8,000 kDa. In further embodiments, the serotype 10A glycoconjugateof the invention has a molecular weight of between 8,000 kDa and 20,000kDa; between 8,000 kDa and 15,000 kDa; or between 8,000 kDa and 10,000kDa.

Any whole number integer within any of the above ranges is contemplatedas an embodiment of the disclosure. The molecular weight of theglycoconjugate is measured by SEC-MALLS.

Another way to characterize the serotype 10A glycoconjugates of theinvention is by the number of lysine residues in the carrier protein(e.g., CRM₁₉₇) that become conjugated to the saccharide which can becharacterized as a range of conjugated lysines (degree of conjugation).The evidence for lysine modification of the carrier protein, due tocovalent linkages to the polysaccharides, can be obtained by amino acidanalysis using routine methods known to those of skill in the art.Conjugation results in a reduction in the number of lysine residuesrecovered compared to the CRM₁₉₇ protein starting material used togenerate the conjugate materials.

In a preferred embodiment, the degree of conjugation of the serotype 10Aglycoconjugate is between 2 and 15, between 2 and 13, between 2 and 10,between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4,between 3 and 15, between 3 and 13, between 3 and 10, between 3 and 8,between 3 and 6, between 3 and 5, between 3 and 4, between 5 and 15,between 5 an 10, between 8 and 15, between 8 and 12, between 10 and 15or between 10 and 12. In a preferred embodiment, the degree ofconjugation of the serotype 10A glycoconjugate is between 6 and 8. In apreferred embodiment, the carrier protein is CRM₁₉₇

The serotype 10A glycoconjugates of the invention may also becharacterized by the ratio (weight/weight) of saccharide to carrierprotein. In some embodiments, the saccharide to carrier protein ratio(w/w) is between 0.5 and 3.0 (e.g., about 0.5, about 0.6, about 0.7,about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0,about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about2.7, about 2.8, about 2.9 or about 3.0). In a preferred embodiment, theratio of serotype 10A saccharide to carrier protein in the conjugate isbetween 0.5 and 2.0, 0.5 and 1.5, 0.5 and 1.0, 1.0 and 1.5 or 1.0 and2.0. In a preferred embodiment, the ratio of serotype 10A polysaccharideto carrier protein in the conjugate is between 0.8 and 1.4. In apreferred embodiment, the ratio of serotype 10A capsular polysaccharideto carrier protein in the conjugate is between 0.8 and 1.2 (e.g., about0.8, about 0.9 about 1.0, about 1.1, or about 1.2). In some suchembodiments, the carrier protein is CRM₁₉₇.

The serotype 10A glycoconjugates and immunogenic compositions of theinvention may contain free saccharide that is not covalently conjugatedto the carrier protein, but is nevertheless present in theglycoconjugate composition. The free saccharide may be noncovalentlyassociated with (i.e., noncovalently bound to, adsorbed to, or entrappedin or with) the glycoconjugate.

In some embodiments, the serotype 10A glycoconjugates of the inventioncomprise less than about 50% free saccharide, less than about 45% freesaccharide, less than about 40% free saccharide, less than about 35%free saccharide, less than about 30% free saccharide, less than about25% free saccharide, less than about 20% free saccharide, less thanabout 15% free saccharide, less than about 10% free saccharide, or lessthan about 5% free saccharide relative to the total amount of 10Asaccharide. Preferably, the serotype 10A glycoconjugate comprises lessthan 15% free saccharide, more preferably less than 10% free saccharide,and still more preferably, less than 5% of free saccharide.

The serotype 10A glycoconjugates may also be characterized by theirmolecular size distribution (K_(d)). Size exclusion chromatography media(CL-4B) can be used to determine the relative molecular sizedistribution of the conjugate, as mentioned above. In a preferredembodiment, at least 30% of the serotype 10A glycoconjugates of theinvention have a K_(d) below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 40% of the serotype 10A glycoconjugatesof the invention have a K_(d) below or equal to 0.3 in a CL-4B column.In a preferred embodiment, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, or 85% of the serotype 10A glycoconjugates of the invention have aK_(d) below or equal to 0.3 in a CL-4B column. In a preferredembodiment, at least 60% of the serotype 10A glycoconjugates have aK_(d) below or equal to 0.3 in a CL-4B column. In a preferredembodiment, between 50% and 80% of the serotype 10A glycoconjugates ofthe invention have a K_(d) below or equal to 0.3 in a CL-4B column.

1.3.7 Glycoconjugates from S. pneumoniae Serotype 11A

In an embodiment, the serotype 11A glycoconjugates are obtained byactivating polysaccharide with 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate (CDAP) to form a cyanate ester. The activatedpolysaccharide may be coupled directly or via a spacer (linker) group toan amino group on the carrier protein. For example, the spacer could becystamine or cysteamine to give a thiolated polysaccharide which couldbe coupled to the carrier via a thioether linkage obtained afterreaction with a maleimide-activated carrier protein (for example usingGMBS) or a haloacetylated carrier protein (for example usingiodoacetimide, SIB, SIAB, sulfo-SIAB, SIA, or SBAP). Preferably, thecyanate ester (optionally made by CDAP chemistry) is coupled with hexanediamine or adipic acid dihydrazide (ADH) and the amino-derivatisedsaccharide is conjugated to the carrier protein using carbodiimide(e.g., EDAC or EDC) chemistry via a carboxyl group on the proteincarrier. Such conjugates are described for example in WO 93/15760, WO95/08348 and WO 96/129094.

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S--NHS, EDC, TSTU.Many are described in International Patent Application Publication No.WO 98/42721. Conjugation may involve a carbonyl linker which may beformed by reaction of a free hydroxyl group of the saccharide with CDI(see Bethell et al. (1979). Biol. Chem. 254:2572-2574; Hearn et al.(1981) J. Chromatogr. 218:509-518) followed by reaction with a proteinto form a carbamate linkage. This may involve reduction of the anomericterminus to a primary hydroxyl group, optional protection/deprotectionof the primary hydroxyl group, reaction of the primary hydroxyl groupwith CDI to form a CDI carbamate intermediate and coupling the CDIcarbamate intermediate with an amino group on a protein.

In preferred embodiments, the serotype 11A glycoconjugates of theinvention are prepared using reductive amination. Reductive aminationinvolves two steps: (1) oxidation of the polysaccharide to generatealdehyde functionalities from vicinal diols in individual hexasaccharideunit and (2) reduction of the activated polysaccharide and a carrierprotein to form a conjugate.

Before oxidation, the serotype 11A polysaccharide is optionallyhydrolized to reduce its viscosity. Mechanical or chemical hydrolysismaybe employed. Chemical hydrolysis maybe conducted using acetic acid.Mechanical sizing maybe conducted using High Pressure HomogenizationShearing.

The oxidation step may involve reaction with periodate. For the purposeof the present invention, the term “periodate” includes both periodateand periodic acid; the term also includes both metaperiodate (IO₄ ⁻) andorthoperiodate (IO₆ ⁵⁻) and the various salts of periodate (e.g., sodiumperiodate and potassium periodate). In an embodiment the capsularpolysaccharidefrom serotype 11A of S. pneumoniae is oxydized in thepresence of metaperiodate, preferably in the presence of sodiumperiodate (NaIO₄). In another embodiment the capsular polysaccharidefrom serotype 11A is oxydized in the presence of orthoperiodate,preferably in the presence of periodic acid.

Following the oxidation step of the polysaccharide, the polysaccharideis said to be activated and is referred to as “activated polysaccharide”here below. The activated polysaccharide maybe purified and lyophilised(freeze-dried).

The activated polysaccharide and the carrier protein may be lyophilized(freeze-dried), either independently (discrete lyophilization) ortogether (co-lyophilized). In one embodiment the activatedpolysaccharide and the carrier protein are co-lyophilized. In anotherembodiment the activated polysaccharide and the carrier protein arelyophilized independently.

In one embodiment the lyophilization takes place in the presence of anon-reducing sugar, possible non-reducing sugars include sucrose,trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitoland palatinit.

The second step of the conjugation process is the reduction of theactivated polysaccharide and a carrier protein to form a conjugate(reductive amination), using a reducing agent. Reducing agents which aresuitable include the cyanoborohydrides, such as sodium cyanoborohydride,borane-pyridine, or borohydride exchange resin. In one embodiment thereducing agent is sodium cyanoborohydride.

In an embodiment, the reduction reaction is carried out in aqueoussolvent, in another embodiment the reaction is carried out in aproticsolvent. In an embodiment, the reduction reaction is carried out in DMSO(dimethylsulfoxide) or in DMF (dimethylformamide) solvent. The DMSO orDMF solvent may be used to reconstitute the activated polysaccharide andcarrier protein which has been lyophilised.

In one embodiment between 0.1 and 3.0, between 0.15 and 2.0, between 0.2and 2.0, or between 0.5 and 1.5 molar equivalents of sodiumcyanoborohydride is used in the reduction reaction. In one embodimentabout 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9 or 3.0 molar equivalents of sodium cyanoborohydride is used in thereduction reaction.

In one embodiment the reducing agent is sodium triacetoxyborohydride. Ina further embodiment between 1.0 and 6.0 molar equivalents, between 2.0and 5.0 molar equivalents or about 3.0 molar equivalents of sodiumtriacetoxyborohydride is used in the reduction reaction.

At the end of the reduction reaction, there may be unreacted aldehydegroups remaining in the conjugates. These may be capped using a suitablecapping agent. In one embodiment this capping agent is sodiumborohydride (NaBH₄). In an embodiment capping is achieved by mixing thereduction reaction with between 0.5 and 5.0 molar equivalents of NaBH4,for example about 1.0, 1.5, 2.0, 2.5 or 3.0 molar equivalents of NaBH₄.

Following the conjugation (the reduction reaction and optionally thecapping), the glycoconjugates may be purified. The glycoconjugates maybe purified by diafiltration and/or ion exchange chromatography and/orsize exclusion chromatography. In an embodiment, the glycoconjugates arepurified by diafiltration or ion exchange chromatography or sizeexclusion chromatography.

In one embodiment the glycoconjugates are sterile filtered.

In some embodiments, the serotype 11A glycoconjugates of the presentinvention are conjugated to the carrier protein (e.g., CRM₁₉₇) andcomprise a saccharide having a molecular weight of between 10 kDa and2,000 kDa. In other such embodiments, the saccharide has a molecularweight of between 50 kDa and 2,000 kDa. In further such embodiments, thesaccharide has a molecular weight of between 50 kDa and 1,750 kDa;between 50 kDa and 1,500 kDa; between 50 kDa and 1,250 kDa; between 50kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDa and 500kDa; between 50 kDa and 400 kDa; between 50 kDa and 300 kDa; between 50kDa and 200 kDa; between 50 kDa and 100 kDa; between 100 kDa and 2,000kDa; between 100 kDa and 1,750 kDa; between 100 kDa and 1,500 kDa;between 100 kDa and 1,250 kDa; between 100 kDa and 1,000 kDa; between100 kDa and 750 kDa; between 100 kDa and 500 kDa; between 100 kDa and400 kDa between; 100 kDa and 300 kDa; between 100 kDa and 200 kDa;between 200 kDa and 2,000 kDa; between 200 kDa and 1,750 kDa; between200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa; between 200 kDaand 1,000 kDa; between 200 kDa and 750 kDa; or between 200 kDa and 500kDa; between 200 kDa and 400 kDa or between 200 kDa and 300 kDa.

In some embodiments, the serotype 11A glycoconjugate of the inventionhas a molecular weight of between 50 kDa and 20,000 kDa. In otherembodiments, the serotype 11A glycoconjugate has a molecular weight ofbetween 50 kDa and 15,000 kDa. In other embodiments, the serotype 11Aglycoconjugate has a molecular weight of between 500 kDa and 10,000 kDa.In other embodiments, the serotype 11A glycoconjugate has a molecularweight of between 200 kDa and 10,000 kDa. In still other embodiments,the serotype 11A glycoconjugate has a molecular weight of between 1,000kDa and 8,000 kDa or between 2,000 kDa and 8,000 kDa.

In further embodiments, the serotype 11A glycoconjugate of the inventionhas a molecular weight of between 200 kDa and 20,000 kDa; between 200kDa and 17,500 kDa; between 200 kDa and 15,000 kDa; between 200 kDa and10,000 kDa; between 200 kDa and 7,500 kDa; between 200 kDa and 5,000kDa; between 200 kDa and 3,000 kDa; between 200 kDa and 2,000 kDa;between 200 kDa and 1,000 kDa; between 500 kDa and 20,000 kDa; between500 kDa and 17,500 kDa; between 500 kDa and 15,000 kDa; between 500 kDaand 12,500 kDa; between 500 kDa and 10,000 kDa; between 500 kDa and7,500 kDa; between 500 kDa and 6,000 kDa; between 500 kDa and 5,000 kDa;between 500 kDa and 4,000 kDa; between 500 kDa and 3,000 kDa; between500 kDa and 2,000 kDa; between 500 kDa and 1,500 kDa; between 500 kDaand 1,000 kDa; between 700 kDa and 20,000 kDa; between 700 kDa and17,500 kDa; between 700 kDa and 15,000 kDa; between 700 kDa and 12,500kDa; between 700 kDa and 10,000 kDa; between 700 kDa and 7,500 kDa;between 700 kDa and 6,000 kDa; between 700 kDa and 5,000 kDa; between700 kDa and 4,500 kDa; between 700 kDa and 4,000 kDa; between 700 kDaand 3,500 kDa; between 700 kDa and 3,000 kDa; between 700 kDa and 2,000kDa; between 700 kDa and 1,500 kDa; between 1,000 kDa and 20,000 kDa;between 1,000 kDa and 17,500 kDa; between 1,000 kDa and 15,000 kDa;between 1,000 kDa and 12,500 kDa; between 1,000 kDa and 10,000 kDa;between 1,000 kDa and 7,500 kDa; between 1,000 kDa and 6,000 kDa;between 1,000 kDa and 5,000 kDa; between 1,000 kDa and 4,000 kDa;between 1,000 kDa and 2,500 kDa; between 2,000 kDa and 20,000 kDa;between 2,000 kDa and 17,500 kDa; between 2,000 kDa and 15,000 kDa;between 2,000 kDa and 12,500 kDa; between 2,000 kDa and 10,000 kDa;between 2,000 kDa and 7,500 kDa; between 2,000 kDa and 6,000 kDa;between 2,000 kDa and 5,000 kDa; between 2,000 kDa and 4,000 kDa; orbetween 2,000 kDa and 3,000 kDa.

In further embodiments, the serotype 11A glycoconjugate of the inventionhas a molecular weight of between 3,000 kDa and 20,000 kDa; between3,000 kDa and 17,500 kDa; between 3,000 kDa and 15,000 kDa; between3,000 kDa and 10,000 kDa; between 3,000 kDa and 7,500 kDa; between 3,000kDa and 5,000 kDa; between 4,000 kDa and 20,000 kDa; between 4,000 kDaand 17,500 kDa; between 4,000 kDa and 15,000 kDa; between 4,000 kDa and12,500 kDa; between 4,000 kDa and 10,000 kDa; between 4,000 kDa and7,500 kDa; between 4,000 kDa and 6,000 kDa; or between 4,000 kDa and5,000 kDa. In further embodiments, the serotype 11A glycoconjugate ofthe invention has a molecular weight of between 5,000 kDa and 20,000kDa; between 5,000 kDa and 17,500 kDa; between 5,000 kDa and 15,000 kDa;between 5,000 kDa and 10,000 kDa or between 5,000 kDa and 7,500 kDa.

In an embodiment, said serotype 11A glycoconjugates are prepared usingreductive amination.

In a preferred embodiment, the serotype 11A glycoconjugate of theinvention comprises at least 0.3, 0.5, 0.6, 1.0, 1.4, 1.8, 2.2, 2.6,3.0, 3.4, 3.8, 4.2, 4.6 or 5.0 mM acetate per mM serotype 11Apolysaccharide. In a preferred embodiment, the serotype 11Aglycoconjugate comprises at least 1.8, 2.2 or 2.6 mM acetate per mMserotype 11A polysaccharide. In an embodiment, the glycoconjugatecomprises at least 0.6 mM acetate per mM serotype 11A polysaccharide. Ina preferred embodiment, the serotype 11A glycoconjugate of the inventioncomprises at least 0.6, 1.0, 1.4, 1.8, 2.2, 2.6, 3.0, 3.4, 3.8, 4.2 or4.6 mM acetate per mM serotype 11A polysaccharide and less than about5.0 mM acetate per mM serotype 11A polysaccharide. In an embodiment, theserotype 11A glycoconjugate of the invention comprises at least 0.6,1.0, 1.4, 1.8, 2.2, 2.6, or 3.0 mM acetate per mM serotype 11Apolysaccharide and less than about 3.4 mM acetate per mM serotype 11Apolysaccharide. In an embodiment, the serotype 11A glycoconjugate of theinvention comprises at least 0.6, 1.0, 1.4, 1.8, 2.2, 2.6, or about 3.0mM acetate per mM serotype 11A polysaccharide and less than about 3.3 mMacetate per mM serotype 11A polysaccharide. Any of the above number iscontemplated as an embodiment of the disclosure.

In a preferred embodiment, the ratio of mM acetate per mM serotype 11Acapsular polysaccharide in the serotype 11A glycoconjugate to mM acetateper mM serotype 11A capsular polysaccharide in the isolatedpolysaccharide is at least 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or0.95. In a preferred embodiment, the ratio of mM acetate per mM serotype11A capsular polysaccharide in the serotype 11A glycoconjugate to mMacetate per mM serotype 11A capsular polysaccharide in the isolatedpolysaccharide is at least 0.7. In a preferred embodiment, the ratio ofmM acetate per mM serotype 11A capsular polysaccharide in the serotype11A glycoconjugate to mM acetate per mM serotype 11A capsularpolysaccharide in the isolated polysaccharide is at least 0.9. In apreferred embodiment, the presence of O-acetyl groups is determined byion-HPLC analysis.

In a preferred embodiment, the ratio of mM acetate per mM serotype 11Acapsular polysaccharide in the serotype 11A glycoconjugate to mM acetateper mM serotype 11A capsular polysaccharide in the activatedpolysaccharide is at least 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or0.95. In a preferred embodiment, the ratio of mM acetate per mM serotype11A capsular polysaccharide in the serotype 11A glycoconjugate to mMacetate per mM serotype 11A capsular polysaccharide in the activatedpolysaccharide is at least 0.7. In a preferred embodiment, the ratio ofmM acetate per mM serotype 11A capsular polysaccharide in the serotype11A glycoconjugate to mM acetate per mM serotype 11A capsularpolysaccharide in the activated polysaccharide is at least 0.9. In apreferred embodiment, the presence of O-acetyl groups is determined byion-HPLC analysis.

In a preferred embodiment, the serotype 11A glycoconjugate of theinvention comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9or 1.0 mM glycerol per mM serotype 11A polysaccharide. In a preferredembodiment, the serotype 11A glycoconjugate comprises at least 0.2, 0.3or 0.4 mM glycerol per mM serotype 11A polysaccharide. In a preferredembodiment, the serotype 11A glycoconjugate of the invention comprisesat least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9 mM glycerol permM serotype 11A polysaccharide and less than about 1.0 mM glycerol permM serotype 11A polysaccharide. In a preferred embodiment, the serotype11A glycoconjugate of the invention comprises at least 0.3, 0.4, 0.5,0.6, or 0.7 mM glycerol per mM serotype 11A polysaccharide and less thanabout 0.8 mM glycerol per mM serotype 11A polysaccharide. Any of theabove number is contemplated as an embodiment of the disclosure.

Another way to characterize the serotype 11A glycoconjugates of theinvention is by the number of lysine residues in the carrier protein(e.g., CRM₁₉₇) that become conjugated to the saccharide which can becharacterized as a range of conjugated lysines (degree of conjugation).

The evidence for lysine modification of the carrier protein, due tocovalent linkages to the polysaccharides, can be obtained by amino acidanalysis using routine methods known to those of skill in the art.Conjugation results in a reduction in the number of lysine residuesrecovered compared to the CRM₁₉₇ protein starting material used togenerate the conjugate materials.

In a preferred embodiment, the degree of conjugation of the serotype 11Aglycoconjugate of the invention is between 1 and 15, between 1 and 13,between 1 and 10, between 1 and 8, between 1 and 6, between 1 and 5,between 1 and 4, between 2 and 15, between 2 and 13, between 2 and 10,between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4,between 5 and 15, between 5 and 10, between 8 and 15, between 8 and 12,between 10 and 15 or between 10 and 12. In an embodiment, the degree ofconjugation of the serotype 11A glycoconjugate of the invention is about1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about9, about 10, about 11, about 12, about 13, about 14 or about 15. In apreferred embodiment, the degree of conjugation of the serotype 11Aglycoconjugate of the invention is between 1 and 6 or between 2 and 5.In some such embodiments, the carrier protein is CRM₁₉₇.

The serotype 11A glycoconjugates of the invention may also becharacterized by the ratio (weight/weight) of saccharide to carrierprotein. In some embodiments, the saccharide to carrier protein ratio(w/w) is between 0.2 and 4.0 (e.g., about 0.2, about 0.3, about 0.4,about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7,about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0,about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about3.7, about 3.8, about 3.9 or about 4.0). In other embodiments, thesaccharide to carrier protein ratio (w/w) is between 0.7 and 2.5,between 0.8 and 2.0, between 0.7 and 2.0, between 0.8 and 1.5, between0.7 and 1.5, 0.7 and 1.4, between 0.8 and 1.4, between 0.7 and 1.45 orbetween 0.8 and 1.45. In further embodiments, the saccharide to carrierprotein ratio (w/w) is between 0.8 and 1.6 (e.g., about 0.8, about 0.9about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5 orabout 1.6).

In some such embodiments, the carrier protein is CRM₁₉₇. In anembodiment, said serotype 11A glycoconjugates are prepared usingreductive amination.

The serotype 11A glycoconjugates and immunogenic compositions of theinvention may contain free saccharide that is not covalently conjugatedto the carrier protein, but is nevertheless present in theglycoconjugate composition. The free saccharide may be noncovalentlyassociated with (i.e., noncovalently bound to, adsorbed to, or entrappedin or with) the glycoconjugate.

In some embodiments, the serotype 11A glycoconjugates of the inventioncomprise less than about 50% of free serotype 11A capsularpolysaccharide compared to the total amount of serotype 11A capsularpolysaccharide, less than about 45% free saccharide, less than about 40%free saccharide, less than about 35% free saccharide, less than about30% free saccharide, less than about 25% free saccharide, less thanabout 20% free saccharide, less than about 15% free saccharide, lessthan about 10% free saccharide, or less than about 5% of free serotype11A capsular polysaccharide compared to the total amount of serotype 11Acapsular polysaccharide. Preferably, the serotype 11A glycoconjugatecomprises less than 15% free saccharide, more preferably less than 10%free saccharide, and still more preferably, less than 5% of freesaccharide.

The serotype 11A glycoconjugates may also be characterized by theirmolecular size distribution (K_(d)). Size exclusion chromatography media(CL-4B) can be used to determine the relative molecular sizedistribution of the conjugate, as mentioned above. In a preferredembodiment, at least 30% of the serotype 11A glycoconjugates of theinvention has a K_(d) below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, or 85% of the serotype 11A glycoconjugates of the invention has aK_(d) below or equal to 0.3 in a CL-4B column. In a preferredembodiment, at least 60% of the serotype 11A glycoconjugates of theinvention has a K_(d) below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 65% of the serotype 11A glycoconjugatesof the invention has a K_(d) below or equal to 0.3 in a CL-4B column.

1.3.8 Glycoconjugates from S. pneumoniae Serotype 8

In an embodiment, the serotype 8 glycoconjugates are obtained byactivating polysaccharide with 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate (CDAP) to form a cyanate ester. The activatedpolysaccharide may be coupled directly or via a spacer (linker) group toan amino group on the carrier protein. For example, the spacer could becystamine or cysteamine to give a thiolated polysaccharide which couldbe coupled to the carrier via a thioether linkage obtained afterreaction with a maleimide-activated carrier protein (for example usingGMBS) or a haloacetylated carrier protein (for example usingiodoacetimide, SIB, SIAB, sulfo-SIAB, SIA, or SBAP). Preferably, thecyanate ester (optionally made by CDAP chemistry) is coupled with hexanediamine or adipic acid dihydrazide (ADH) and the amino-derivatisedsaccharide is conjugated to the carrier protein using carbodiimide(e.g., EDAC or EDC) chemistry via a carboxyl group on the proteincarrier. Such conjugates are described for example in WO 93/15760, WO95/08348 and WO 96/129094.

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S--NHS, EDC, TSTU.Many are described in International Patent Application Publication No.WO 98/42721. Conjugation may involve a carbonyl linker which may beformed by reaction of a free hydroxyl group of the saccharide with CDI(see Bethell et al. (1979) J. Biol. Chem. 254:2572-2574; Hearn et al.(1981) J. Chromatogr. 218:509-518) followed by reaction with a proteinto form a carbamate linkage. This may involve reduction of the anomericterminus to a primary hydroxyl group, optional protection/deprotectionof the primary hydroxyl group, reaction of the primary hydroxyl groupwith CDI to form a CDI carbamate intermediate and coupling the CDIcarbamate intermediate with an amino group on a protein.

In preferred embodiments, the serotype 8 glycoconjugates of theinvention are prepared using reductive amination. Reductive aminationinvolves two steps: (1) oxidation of the polysaccharide to generatealdehyde functionalities from vicinal diols in individual hexasaccharideunit and (2) reduction of the activated polysaccharide and a carrierprotein to form a conjugate.

Before oxidation, the serotype 8 polysaccharide is optionally hydrolizedto reduce its viscosity. Mechanical or chemical hydrolysis maybeemployed. Chemical hydrolysis maybe conducted using acetic acid.

The oxidation step may involve reaction with periodate. For the purposeof the present invention, the term “periodate” includes both periodateand periodic acid; the term also includes both metaperiodate (IO₄ ⁻) andorthoperiodate (IO₆ ⁵⁻) and the various salts of periodate (e.g., sodiumperiodate and potassium periodate). In an embodiment the capsularpolysaccharidefrom serotype 8 of S. pneumoniae is oxydized in thepresence of metaperiodate, preferably in the presence of sodiumperiodate (NaIO₄). In another embodiment the capsular polysaccharidefrom serotype 8 is oxydized in the presence of orthoperiodate,preferably in the presence of periodic acid.

Following the oxidation step of the polysaccharide, the polysaccharideis said to be activated and is referred to as “activated polysaccharide”here below. The activated polysaccharide maybe purified and lyophilised(freeze-dried).

The activated polysaccharide and the carrier protein may be lyophilised(freeze-dried), either independently (discrete lyophilization) ortogether (co-lyophilized). In one embodiment the activatedpolysaccharide and the carrier protein are co-lyophilised. In anotherembodiment the activated polysaccharide and the carrier protein arelyophilised independently.

In one embodiment the lyophilisation takes place in the presence of anon-reducing sugar, possible non-reducing sugars include sucrose,trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitoland palatinit.

The second step of the conjugation process is the reduction of theactivated polysaccharide and a carrier protein to form a conjugate(reductive amination), using a reducing agent. Reducing agents which aresuitable include the cyanoborohydrides, such as sodium cyanoborohydride,borane-pyridine, or borohydride exchange resin. In one embodiment thereducing agent is sodium cyanoborohydride.

In an embodiment, the reduction reaction is carried out in aqueoussolvent, in another embodiment the reaction is carried out in aproticsolvent. In an embodiment, the reduction reaction is carried out in DMSO(dimethylsulfoxide) or in DMF (dimethylformamide) solvent. The DMSO orDMF solvent may be used to reconstitute the activated polysaccharide andcarrier protein which has been lyophilised.

In one embodiment between 0.1 and 3.0, between 0.15 and 2.0, between 0.2and 1.0, or between 0.25 and 0.5 molar equivalents of sodiumcyanoborohydride is used in the reduction reaction. In one embodimentabout 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9 or 3.0 molar equivalents of sodium cyanoborohydride is used in thereduction reaction.

In one embodiment the reducing agent is sodium triacetoxyborohydride. Ina further embodiment between 1.0 and 6.0 molar equivalents, between 2.0and 5.0 molar equivalents or about 3.0 molar equivalents of sodiumtriacetoxyborohydride is used in the reduction reaction.

At the end of the reduction reaction, there may be unreacted aldehydegroups remaining in the conjugates, these may be capped using a suitablecapping agent. In one embodiment this capping agent is sodiumborohydride (NaBH₄). In an embodiment capping is achieved by mixing thereduction reaction with between 0.5 and 5.0 molar equivalents of NaBH₄,for example about 1.0, 1.5, 2.0, 2.5 or 3.0 molar equivalents of NaBH₄.

Following the conjugation (the reduction reaction and optionally thecapping), the glycoconjugates may be purified. The glycoconjugates maybepurified by diafiltration and/or ion exchange chromatography and/or sizeexclusion chromatography. In an embodiment, the glycoconjugates arepurified by diafiltration or ion exchange chromatography or sizeexclusion chromatography.

In one embodiment the glycoconjugates are sterile filtered.

In some embodiments, the serotype 8 glycoconjugates of the presentinvention are conjugated to the carrier protein (e.g., CRM₁₉₇) andcomprise a saccharide having a molecular weight of between 10 kDa and2,000 kDa. In other such embodiments, the saccharide has a molecularweight of between 50 kDa and 2,000 kDa. In further such embodiments, thesaccharide has a molecular weight of between 50 kDa and 1,750 kDa;between 50 kDa and 1,500 kDa; between 50 kDa and 1,250 kDa; between 50kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDa and 500kDa; between 100 kDa and 2,000 kDa; between 100 kDa and 1,750 kDa;between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa; between100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100 kDa and500 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and 1,750 kDa;between 200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa; between200 kDa and 1,000 kDa; between 200 kDa and 750 kDa; or between 200 kDaand 500 kDa; or between 200 kDa and 400 kDa. In an embodiment, saidserotype 8 glycoconjugates are prepared using reductive amination.

In some embodiments, the serotype 8 glycoconjugate of the invention hasa molecular weight of between 50 kDa and 20,000 kDa. In otherembodiments, the serotype 8 glycoconjugate has a molecular weight ofbetween 50 kDa and 15,000 kDa. In other embodiments, the serotype 8glycoconjugate has a molecular weight of between 500 kDa and 10,000 kDa.In other embodiments, the serotype 8 glycoconjugate has a molecularweight of between 200 kDa and 10,000 kDa. In still other embodiments,the the serotype 8 glycoconjugate has a molecular weight of between1,000 kDa and 8,000 kDa or between 2,000 kDa and 8,000 kDa.

In further embodiments, the serotype 8 glycoconjugate of the inventionhas a molecular weight of between 200 kDa and 20,000 kDa; between 200kDa and 15,000 kDa; between 200 kDa and 10,000 kDa; between 200 kDa and7,500 kDa; between 200 kDa and 5,000 kDa; between 200 kDa and 3,000 kDa;between 200 kDa and 1,000 kDa; between 500 kDa and 20,000 kDa; between500 kDa and 15,000 kDa; between 500 kDa and 12,500 kDa; between 500 kDaand 10,000 kDa; between 500 kDa and 7,500 kDa; between 500 kDa and 6,000kDa; between 500 kDa and 5,000 kDa; between 500 kDa and 4,000 kDa;between 500 kDa and 3,000 kDa; between 500 kDa and 2,000 kDa; between500 kDa and 1,500 kDa; between 500 kDa and 1,000 kDa; between 750 kDaand 20,000 kDa; between 750 kDa and 15,000 kDa; between 750 kDa and12,500 kDa; between 750 kDa and 10,000 kDa; between 750 kDa and 7,500kDa; between 750 kDa and 6,000 kDa; between 750 kDa and 5,000 kDa;between 750 kDa and 4,000 kDa; between 750 kDa and 3,000 kDa; between750 kDa and 2,000 kDa; between 750 kDa and 1,500 kDa; between 1,000 kDaand 15,000 kDa; between 1,000 kDa and 12,500 kDa; between 1,000 kDa and10,000 kDa; between 1,000 kDa and 7,500 kDa; between 1,000 kDa and 6,000kDa; between 1,000 kDa and 5,000 kDa; between 1,000 kDa and 4,000 kDa;between 1,000 kDa and 2,500 kDa; between 2,000 kDa and 15,000 kDa;between 2,000 kDa and 12,500 kDa; between 2,000 kDa and 10,000 kDa;between 2,000 kDa and 7,500 kDa; between 2,000 kDa and 6,000 kDa;between 2,000 kDa and 5,000 kDa; between 2,000 kDa and 4,000 kDa; orbetween 2,000 kDa and 3,000 kDa.

In further embodiments, the serotype 8 glycoconjugate of the inventionhas a molecular weight of between 3,000 kDa and 20,000 kDa; between3,000 kDa and 15,000 kDa; between 3,000 kDa and 10,000 kDa; between3,000 kDa and 7,500 kDa; between 3,000 kDa and 5,000 kDa; between 4,000kDa and 20,000 kDa; between 4,000 kDa and 15,000 kDa; between 4,000 kDaand 12,500 kDa; between 4,000 kDa and 10,000 kDa; between 4,000 kDa and7,500 kDa; between 4,000 kDa and 6,000 kDa; or between 4,000 kDa and5,000 kDa. In further embodiments, the serotype 8 glycoconjugate of theinvention has a molecular weight of between 5,000 kDa and 20,000 kDa;between 5,000 kDa and 15,000 kDa; between 5,000 kDa and 10,000 kDa orbetween 5,000 kDa and 7,500 kDa. In further embodiments, the serotype 8glycoconjugate of the invention has a molecular weight of between 6,000kDa and 20,000 kDa; between 6,000 kDa and 15,000 kDa; between 6,000 kDaand 10,000 kDa or between 6,000 kDa and 7,500 kDa. In furtherembodiments, the serotype 8 glycoconjugate of the invention has amolecular weight of between 7,000 kDa and 20,000 kDa; between 7,000 kDaand 15,000 kDa; between 7,000 kDa and 10,000 kDa or between 7,000 kDaand 8,000 kDa. In further embodiments, the serotype 8 glycoconjugate ofthe invention has a molecular weight of between 8,000 kDa and 20,000kDa; between 8,000 kDa and 15,000 kDa; or between 8,000 kDa and 10,000kDa.

In an embodiment, said serotype 8 glycoconjugates are prepared usingreductive amination.

Another way to characterize the serotype 8 glycoconjugates of theinvention is by the number of lysine residues in the carrier protein(e.g., CRM₁₉₇) that become conjugated to the saccharide which can becharacterized as a range of conjugated lysines (degree of conjugation).

The evidence for lysine modification of the carrier protein, due tocovalent linkages to the polysaccharides, can be obtained by amino acidanalysis using routine methods known to those of skill in the art. Infrequent embodiments, the carrier protein is covalently conjugated toactivated polysaccharide through an amine linkage to one or more ε-aminogroups of lysine residues on the carrier protein. In some suchembodiments, the carrier protein comprises 2 to 20 lysine residuescovalently conjugated to the saccharide. In other such embodiments, thecarrier protein comprises 4 to 16 or 6 to 14 lysine residues covalentlyconjugated to the saccharide.

In a preferred embodiment, the degree of conjugation of the serotype 8glycoconjugate of the invention is between 2 and 20, between 2 and 15,between 2 and 13, between 2 and 10, between 2 and 8, between 2 and 6,between 2 and 5, between 2 and 4, between 3 and 15, between 3 and 13,between 3 and 10, between 3 and 8, between 3 and 6, between 3 and 5,between 3 and 4, between 5 and 15, between 5 and 10, between 8 and 15,between 8 and 12, between 10 and 15 or between 10 and 12. In anembodiment, the degree of conjugation of the serotype 8 glycoconjugateof the invention is about 2, about 3, about 4, about 5, about 6, about7, about 8, about 9, about 10, about 11, about 12, about 13, about 14 orabout 15. In a preferred embodiment, the degree of conjugation of theserotype 8 glycoconjugate of the invention is between 4 and 16 orbetween 6 and 14. In some such embodiments, the carrier protein isCRM₁₉₇. In a preferred embodiment, the carrier protein comprises CRM₁₉₇,which contains 39 lysine residues. In some such embodiments, the CRM₁₉₇may comprise between 4 and 16 or between 6 and 14 lysine residues out of39 covalently linked to the saccharide. Another way to express thisparameter is that about 10% to about 41% or about 15% to about 36% ofCRM₁₉₇ lysines are covalently linked to the saccharide. In another suchembodiment, the CRM₁₉₇ may comprise 2 to 20 lysine residues out of 39covalently linked to the saccharide. Another way to express thisparameter is that about 5% to about 50% of CRM₁₉₇ lysines are covalentlylinked to the saccharide. In some such embodiments, the CRM₁₉₇ maycomprise about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 lysineresidues out of 39 covalently linked to the saccharide.

The serotype 8 glycoconjugates of the invention may also becharacterized by the ratio (weight/weight) of saccharide to carrierprotein. In some embodiments, the saccharide to carrier protein ratio(w/w) is between 0.2 and 4.0 (e.g., about 0.2, about 0.3, about 0.4,about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7,about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0,about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about3.7, about 3.8, about 3.9 or about 4.0). In other embodiments, thesaccharide to carrier protein ratio (w/w) is between 0.7 and 2.5. Infurther embodiments, the saccharide to carrier protein ratio (w/w) isbetween 0.8 and 1.5 (e.g., about 0.8, about 0.9 about 1.0, about 1.1,about 1.2, about 1.3, about 1.4 or about 1.5). In some such embodiments,the carrier protein is CRM₁₉₇. In an embodiment, said serotype 8glycoconjugates are prepared using reductive amination.

The serotype 8 glycoconjugates and immunogenic compositions of theinvention may contain free saccharide that is not covalently conjugatedto the carrier protein, but is nevertheless present in theglycoconjugate composition. The free saccharide may be noncovalentlyassociated with (i.e., noncovalently bound to, adsorbed to, or entrappedin or with) the glycoconjugate.

In some embodiments, the serotype 8 glycoconjugates of the inventioncomprise less than about 50% free saccharide, less than about 45% freesaccharide, less than about 40% free saccharide, less than about 35%free saccharide, less than about 30% free saccharide, less than about25% free saccharide, less than about 20% free saccharide, less thanabout 15% free saccharide, less than about 10% free saccharide, or lessthan about 5% free saccharide relative to the total amount of serotype 8saccharide. Preferably, the serotype 8 glycoconjugate comprises lessthan 15% free saccharide, more preferably less than 10% free saccharide,and still more preferably, less than 5% of free saccharide.

The serotype 8 glycoconjugates may also be characterized by theirmolecular size distribution (K_(d)). Size exclusion chromatography media(CL-4B) can be used to determine the relative molecular sizedistribution of the conjugate. Size Exclusion Chromatography (SEC) isused in gravity fed columns to profile the molecular size distributionof conjugates. Large molecules excluded from the pores in the mediaelute more quickly than small molecules. Fraction collectors are used tocollect the column eluate. The fractions are tested colorimetrically bysaccharide assay. For the determination of K_(d), columns are calibratedto establish the fraction at which molecules are fully excluded (V₀),(K_(d)=0), and the fraction representing the maximum retention (V_(i)),(K_(d)=1). The fraction at which a specified sample attribute is reached(V_(e)), is related to K_(d) by the expression,K_(d)=(V_(e)−V₀)/(V_(i)−V₀).

In a preferred embodiment, at least 40% of the serotype 8glycoconjugates of the invention have a K_(d) below or equal to 0.3 in aCL-4B column. In a preferred embodiment, at least 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the serotype 8glycoconjugates of the invention have a K_(d) below or equal to 0.3 in aCL-4B column. In a preferred embodiment, at least 60% of the serotype 8glycoconjugates of the invention have a K_(d) below or equal to 0.3 in aCL-4B column. In a preferred embodiment, at least 70% of the serotype 8glycoconjugates of the invention have a K_(d) below or equal to 0.3 in aCL-4B column.

In a preferred embodiment, between 40% and 90% of the serotype 8glycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column. Ina preferred embodiment, between 50% and 90% of the serotype 8glycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column. Ina preferred embodiment, between 65% and 80% of the serotype 8glycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column.

2. IMMUNOGENIC COMPOSITIONS OF THE PRESENT INVENTION

In an embodiment, the number of S. pneumoniae capsular saccharides ofthe immunogenic composition can range from 1 serotype (or “v”, valence)to 7 different serotypes (7v). In one embodiment there is 1 serotype. Inone embodiment there are 2 different serotypes. In one embodiment thereare 3 different serotypes. In one embodiment there are 4 differentserotypes. In one embodiment there are 5 different serotypes. In oneembodiment there are 6 different serotypes. In one embodiment there are7 different serotypes. The capsular saccharides are conjugated to acarrier protein to form glycoconjugates as described here above.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate selected from the group consisting of aglycoconjugate from S. pneumoniae serotype 15B (such as theglycoconjugates of section 1.3.4 above), a glycoconjugate from S.pneumoniae serotype 22F (such as the glycoconjugates of section 1.3.2above), a glycoconjugate from S. pneumoniae serotype 33F (such as theglycoconjugates of section 1.3.3 above), a glycoconjugate from S.pneumoniae serotype 12F (such as the glycoconjugates of section 1.3.5above), a glycoconjugate from S. pneumoniae serotype 10A (such as theglycoconjugates of section 1.3.6 above), a glycoconjugate from S.pneumoniae serotype 11A (such as the glycoconjugates of section 1.3.7above) and a glycoconjugate from S. pneumoniae serotype 8 (such as theglycoconjugates of section 1.3.8 above).

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate from S. pneumoniae serotype 15B, such as theglycoconjugate of section 1.3.4 above. In an embodiment the immunogeniccomposition of the invention comprises at least one glycoconjugate fromS. pneumoniae serotype 22F, such as the ones disclosed at section 1.3.2above. In an embodiment the immunogenic composition of the inventioncomprises at least one glycoconjugate from S. pneumoniae serotype 33Fsuch as the ones disclosed at section 1.3.3 above. In an embodiment theimmunogenic composition of the invention comprises at least oneglycoconjugate from S. pneumoniae serotype 12F such as the onesdisclosed at section 1.3.5 above. In an embodiment the immunogeniccomposition of the invention comprises at least one glycoconjugate fromS. pneumoniae serotype 10A such as the ones disclosed at section 1.3.6above. In an embodiment the immunogenic composition of the inventioncomprises at least one glycoconjugate from S. pneumoniae serotype 11Asuch as the ones disclosed at section 1.3.7 above. In an embodiment theimmunogenic composition of the invention comprises at least oneglycoconjugate from S. pneumoniae serotype 8 such as the ones disclosedat section 1.3.8 above.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the two S. pneumoniae serotypesselected from the group consisting of: 15B and 22F, 15B and 33F, 15B and12F, 15B and 10A, 15B and 11A, 15B and 8, 22F and 33F, 22F and 12F, 22Fand 10A, 22F and 11A, 22F and 8, 33F and 12F, 33F and 10A, 33F and 11A,33F and 8, 12F and 10A, 12F and 11A, 12F and 8, 10A and 11A, 10A and 8,and 11A and 8.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the three following S. pneumoniaeserotypes:

15B and 22F and 33F, 15B and 22F and 12F, 15B and 22F and 10A, 15B and22F and 11A, 15B and 22F and 8, 15B and 33F and 12F, 15B and 33F and10A, 15B and 33F and 11A, 15B and 33F and 8, 15B and 12F and 10A, 15Band 12F and 11A, 15B and 12F and 8, 15B and 10A and 11A, 15B and 10A and8, 15B and 11A and 8, 22F and 33F and 12F, 22F and 33F and 10A, 22F and33F and 11A, 22F and 33F and 8, 22F and 12F and 10A, 22F and 12F and11A, 22F and 12F and 8, 22F and 10A and 11A, 22F and 10A and 8, 22F and11A and 8, 33F and 12F and 10A, 33F and 12F and 11A, 33F and 12F and 8,33F and 10A and 11A, 33F and 10A and 8, 33F and 11A and 8, 12F and 10Aand 11A, 12F and 10A and 8, 12F and 11A and 8, or 10A and 11A and 8.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the four following S. pneumoniaeserotypes:

15B and 22F and 33F and 12F, 15B and 22F and 33F and 10A, 15B and 22Fand 33F and 11A, 15B and 22F and 33F and 8, 15B and 22F and 12F and 10A,15B and 22F and 12F and 11A, 15B and 22F and 12F and 8, 15B and 22F and10A and 11A, 15B and 22F and 10A and 8, 15B and 22F and 11A and 8, 15Band 33F and 12F and 10A, 15B and 33F and 12F and 11A, 15B and 33F and12F and 8, 15B and 33F and 10A and 11A, 15B and 33F and 10A and 8, 15Band 33F and 11A and 8, 15B and 12F and 10A and 11A, 15B and 12F and 10Aand 8, 15B and 12F and 11A and 8, 15B and 10A and 11A and 8, 22F and 33Fand 12F and 10A, 22F and 33F and 12F and 11A, 22F and 33F and 12F and 8,22F and 33F and 10A and 11A, 22F and 33F and 10A and 8, 22F and 33F and11A and 8, 22F and 12F and 10A and 11A, 22F and 12F and 10A and 8, 22Fand 12F and 11A and 8, 22F and 10A and 11A and 8, 33F and 12F and 10Aand 11A, 33F and 12F and 10A and 8, 33F and 12F and 11A and 8, 33F and10A and 11A and 8 or 12F and 10A and 11A and 8.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the five following S. pneumoniaeserotypes:

15B and 22F and 33F and 12F and 10A, 15B and 22F and 33F and 12F and11A, 15B and 22F and 33F and 12F and 8, 15B and 22F and 33F and 10A and11A, 15B and 22F and 33F and 10A and 8, 15B and 22F and 33F and 11A and8, 15B and 22F and 12F and 10A and 11A, 15B and 22F and 12F and 10A and8, 15B and 22F and 12F and 11A and 8, 15B and 22F and 10A and 11A and 8,15B and 33F and 12F and 10A and 11A, 15B and 33F and 12F and 10A and 8,15B and 33F and 12F and 11A and 8, 15B and 33F and 10A and 11A and 8,15B and 12F and 10A and 11A and 8, 22F and 33F and 12F and 10A and 11A,22F and 33F and 12F and 10A and 8, 22F and 33F and 12F and 11A and 8,22F and 33F and 10A and 11A and 8, 22F and 12F and 10A and 11A and 8 or33F and 12F and 10A and 11A and 8.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the six following S. pneumoniaeserotypes:

15B and 22F and 33F and 12F and 10A and 11A, 15B and 22F and 33F and 12Fand 10A and 8, 15B and 22F and 33F and 12F and 11A and 8, 15B and 22Fand 33F and 10A and 11A and 8, 15B and 22F and 12F and 10A and 11A and8, 15B and 33F and 12F and 10A and 11A and 8 or 22F and 33F and 12F and10A and 11A and 8.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the seven following S. pneumoniaeserotypes: 15B and 22F and 33F and 12F and 10A and 11A and 8.

In an embodiment the glycoconjugates from S. pneumoniae serotypes 15B,22F, 33F, 12F, 10A, 11A and/or 8 of any of the immunogenic compositiondefined in this section are as disclosed at sections 1.3.2 to 1.3.8above.

Preferably, all the glycoconjugates of the above immunogeniccompositions are individually conjugated to the carrier protein.

In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 22F is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 33F is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 15B is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 12F is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 10A is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 11A is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 8 is conjugated to CRM₁₉₇.

In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae are all individually conjugated toCRM₁₉₇.

In another embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae are all individually conjugated toPD. In another embodiment, the glycoconjugates from S. pneumoniae areall individually conjugated to TT. In yet another embodiment, theglycoconjugates from S. pneumoniae are all individually conjugated toDT.

In another embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 22F, 33F, 15B, 12F, 10A,11A, and/or 8 is/are individually conjugated to DT. In anotherembodiment, the glycoconjugates from S. pneumoniae serotype 22F, 33F,15B, 12F, 10A, 11A, and/or 8 is/are individually conjugated to TT. Inanother embodiment, the glycoconjugates from S. pneumoniae serotype 22F,33F, 15B, 12F, 10A, 11A, and/or 8 is/are individually conjugated to PD.

In another embodiment of any of the above immunogenic compositions, atleast one of the glycoconjugates is individually conjugated to DT andthe other glycoconjugate(s) from S. pneumoniae is/are individuallyconjugated to TT. In another embodiment, at least one of theglycoconjugates is individually conjugated to TT and the otherglycoconjugate(s) is/are individually conjugated to DT. In anotherembodiment, at least one of the glycoconjugates is individuallyconjugated to PD and the other glycoconjugate(s) is/are individuallyconjugated to DT. In another embodiment, at least one of theglycoconjugates is individually conjugated to PD and the otherglycoconjugate(s) is/are individually conjugated to TT. In anotherembodiment, at least one of the glycoconjugates is individuallyconjugated to TT and the other glycoconjugate(s) is/are individuallyconjugated to PD. In another embodiment, at least one of theglycoconjugates is individually conjugated to DT and the otherglycoconjugate(s) is/are individually conjugated to PD.

In another embodiment of any of the above immunogenic compositions, atleast one of the glycoconjugates is individually conjugated to CRM₁₉₇and the other glycoconjugate(s) from S. pneumoniae is/are individuallyconjugated to DT. In another embodiment, at least one of theglycoconjugates is individually conjugated to CRM₁₉₇ and the otherglycoconjugate(s) is/are individually conjugated to TT. In anotherembodiment, at least one of the glycoconjugates is individuallyconjugated to CRM₁₉₇ and the other glycoconjugate(s) is/are individuallyconjugated to PD. In another embodiment, at least one of theglycoconjugates is individually conjugated to DT and the otherglycoconjugate(s) is/are individually conjugated to CRM₁₉₇. In anotherembodiment, at least one of the glycoconjugates is individuallyconjugated to TT and the other glycoconjugate(s) is/are individuallyconjugated to CRM₁₉₇. In another embodiment, at least one of theglycoconjugates is individually conjugated to PD and the otherglycoconjugate(s) is/are individually conjugated to CRM₁₉₇.

In an embodiment the above immunogenic compositions comprise from 1 to 7different serotypes of S. pneumoniae. In one embodiment the aboveimmunogenic composition is a 1, 2, 3, 4, 5, 6 or 7-valent pneumococcalconjugate composition. In one embodiment the above immunogeniccomposition is a 6-valent pneumococcal conjugate composition. In oneembodiment the above immunogenic composition is a 7-valent pneumococcalconjugate composition.

1. In an embodiment the immunogenic composition of the inventioncomprises at least one glycoconjugate from S. pneumoniae serotype 15B,such as the glycoconjugates of section 1.3.4 above.2. In another embodiment the immunogenic composition of the inventioncomprises in addition to point 1 above, at least one glycoconjugate fromS. pneumoniae serotype 22F, such as the ones disclosed at section 1.3.2above.3. In another embodiment the immunogenic composition of the inventioncomprises in addition to point 1 or 2 above, at least one glycoconjugatefrom S. pneumoniae serotype 33F such as the ones disclosed at section1.3.3 above.4. In another embodiment the immunogenic composition of the inventioncomprises in addition to point 1, 2 or 3 above, at least oneglycoconjugate from S. pneumoniae serotype 12F such as the onesdisclosed at section 1.3.5 above.5. In another embodiment the immunogenic composition of the inventioncomprises in addition to point 1, 2, 3 or 4 above, at least oneglycoconjugate from S. pneumoniae serotype 10A such as the onesdisclosed at section 1.3.6 above.6. In another embodiment the immunogenic composition of the inventioncomprises in addition to point 1, 2, 3, 4 or 5 above, at least oneglycoconjugate from S. pneumoniae serotype 11A such as the onesdisclosed at section 1.3.7 above.7. In another embodiment the immunogenic composition of the inventioncomprises in addition to point 1, 2, 3, 4, 5 or 6 above, at least oneglycoconjugate from S. pneumoniae serotype 8 such as the ones disclosedat section 1.3.8 above.

In an embodiment, the immunogenic composition of the invention comprisesconjugated S. pneumoniae saccharides from serotypes 8, 10A, 11A, 12F,15B, 22F and 33F.

In an embodiment, the glycoconjugates of the immunogenic composition ofthe invention consist of glycoconjugates from S. pneumoniae serotypes 8,10A, 11A, 12F, 15B, 22F and 33F.

Preferably, all the glycoconjugates of the immunogenic composition ofthe invention (e.g., of any of points 1 to 7 above) are individuallyconjugated to the carrier protein.

In an embodiment of any of points 1 to 7 above, the glycoconjugate fromS. pneumoniae serotype 22F is conjugated to CRM₁₉₇. In an embodiment ofany of points 2 to 7 above, the glycoconjugate from S. pneumoniaeserotype 33F is conjugated to CRM₁₉₇. In an embodiment of any of points3 to 7 above, the glycoconjugate from S. pneumoniae serotype 15B isconjugated to CRM₁₉₇. In an embodiment of any of points 4 to 7 above,the glycoconjugate from S. pneumoniae serotype 12F is conjugated toCRM₁₉₇. In an embodiment of any of points 5 to 7 above, theglycoconjugate from S. pneumoniae serotype 10A is conjugated to CRM₁₉₇.In an embodiment of any of points 6 to 7 above, the glycoconjugate fromS. pneumoniae serotype 11A is conjugated to CRM₁₉₇. In an embodiment ofpoint 7 above, the glycoconjugate from S. pneumoniae serotype 8 isconjugated to CRM₁₉₇.

In an embodiment, the glycoconjugates of the immunogenic composition ofpoints 1 to 7 above are individually conjugated to CRM₁₉₇.

In an embodiment, the glycoconjugates of the immunogenic composition ofpoints 1 to 7 above are individually conjugated to PD. In an embodiment,the glycoconjugates of the immunogenic composition of points 1 to 7above are individually conjugated to TT. In an embodiment, theglycoconjugates of the immunogenic composition of points 1 to 7 aboveare individually conjugated to DT.

In an embodiment, at least one of the glycoconjugates of the immunogeniccomposition of points 1 to 7 above is individually conjugated to DT andthe other glycoconjugate(s) from S. pneumoniae is/are individuallyconjugated to TT. In another embodiment, at least one of theglycoconjugates of the immunogenic composition of points 1 to 7 above isindividually conjugated to TT and the other glycoconjugate(s) is/areindividually conjugated to DT. In another embodiment, at least one ofthe glycoconjugates of the immunogenic composition of points 1 to 7above is individually conjugated to PD and the other glycoconjugate(s)is/are individually conjugated to DT. In another embodiment, at leastone of the glycoconjugates of the immunogenic composition of points 1 to7 above is individually conjugated to PD and the other glycoconjugate(s)is/are individually conjugated to TT. In another embodiment, at leastone of the glycoconjugates of the immunogenic composition of points 1 to7 above is individually conjugated to TT and the other glycoconjugate(s)is/are individually conjugated to PD. In another embodiment, at leastone of the glycoconjugates of the immunogenic composition of points 1 to7 above is individually conjugated to DT and the other glycoconjugate(s)is/are individually conjugated to PD.

In another embodiment, at least one of the glycoconjugates of theimmunogenic composition of points 1 to 7 above is individuallyconjugated to CRM₁₉₇ and the other glycoconjugate(s) from S. pneumoniaeis/are individually conjugated to DT. In another embodiment, at leastone of the glycoconjugates of the immunogenic composition of points 1 to7 above is individually conjugated to CRM₁₉₇ and the otherglycoconjugate(s) is/are individually conjugated to TT. In anotherembodiment, at least one of the glycoconjugates of the immunogeniccomposition of points 1 to 7 above is individually conjugated to CRM₁₉₇and the other glycoconjugate(s) is/are individually conjugated to PD. Inanother embodiment, at least one of the glycoconjugates of theimmunogenic composition of points 1 to 7 above is individuallyconjugated to DT and the other glycoconjugate(s) is/are individuallyconjugated to CRM₁₉₇. In another embodiment, at least one of theglycoconjugates of the immunogenic composition of points 1 to 7 above isindividually conjugated to TT and the other glycoconjugate(s) is/areindividually conjugated to CRM₁₉₇. In another embodiment, at least oneof the glycoconjugates of the immunogenic composition of points 1 to 7above is individually conjugated to PD and the other glycoconjugate(s)is/are individually conjugated to CRM₁₉₇.

In an embodiment the above immunogenic composition is a 1, 2, 3, 4, 5, 6or 7-valent pneumococcal conjugate composition. In one embodiment theabove immunogenic composition is a 6-valent pneumococcal conjugatecomposition. In one embodiment the above immunogenic composition is a7-valent pneumococcal conjugate composition.

After conjugation of the capsular polysaccharide to the carrier protein,the glycoconjugates are purified (enriched with respect to the amount ofpolysaccharide-protein conjugate) by a variety of techniques. Thesetechniques include concentration/diafiltration operations,precipitation/elution, column chromatography, and depth filtration (seefor example U.S. Patent App. Pub. No. 2007/0184072 or WO 2008/079653).After the individual glycoconjugates are purified, they are compoundedto formulate the immunogenic composition of the present invention.

In an embodiment the dosage of the above immunogenic composition is asdisclosed at section 5 below.

In an embodiment the above immunogenic compositions further compriseantigen(s) from other pathogens, particularly from bacteria and/orviruses such as disclosed at section 6 below.

In an embodiment the above immunogenic compositions further comprise oneor more adjuvants as disclosed at section 6 below.

In an embodiment the above immunogenic compositions are formulated asdisclosed at section 8 below.

3. IMMUNOGENIC COMPOSITIONS WHICH MAY BE USED IN COMBINATION WITH THEIMMUNOGENIC COMPOSITIONS OF THE PRESENT INVENTION

In an embodiment, the immunogenic compositions of the invention (such asany of the ones of section 2 above) are used in combination with asecond immunogenic composition.

In an embodiment, said second immunogenic composition comprises at leastone glycoconjugate from a Streptococcus pneumoniae serotype selectedfrom the group consisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F, 22F and 33F.

In an embodiment, said second immunogenic composition comprises at leastone glycoconjugate from a Streptococcus pneumoniae serotype selectedfrom the group consisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F.

1. In an embodiment said second immunogenic composition comprises atleast one glycoconjugate from S. pneumoniae serotypes 4, 6B, 9V, 14,18C, 19F and 23F (such as the glycoconjugates of section 1.3.1 above).2. In another embodiment said second immunogenic composition comprisesin addition to point 1 above, at least one glycoconjugate from S.pneumoniae serotypes 1, 5 and 7F (such as the glycoconjugates of section1.3.1 above).3. In another embodiment said second immunogenic composition comprisesin addition to point 1 or 2 above, at least one glycoconjugate from S.pneumoniae serotypes 6A and 19A (such as the glycoconjugates of section1.3.1 above).4. In another embodiment said second immunogenic composition comprisesin addition to point 1, 2 or 3 above, at least one glycoconjugate fromS. pneumoniae serotype 3 (such as the glycoconjugates of section 1.3.1above).5. In another embodiment said second immunogenic composition comprisesin addition to point 1, 2, 3 or 4 above, at least one glycoconjugatefrom S. pneumoniae serotype 22F, such as the ones disclosed at section1.3.2 above.6. In another embodiment said second immunogenic composition comprisesin addition to point 1, 2, 3, 4 or 5 above, at least one glycoconjugatefrom S. pneumoniae serotype 33F such as the ones disclosed at section1.3.3 above.

Preferably, all the glycoconjugates of the above second immunogeniccompositions are individually conjugated to the carrier protein.

In an embodiment of any of the above second immunogenic compositions,the glycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19Fand 23F are conjugated to CRM₁₉₇. In an embodiment of any of the abovesecond immunogenic compositions, the glycoconjugates from S. pneumoniaeserotypes 1, 5 and 7F are conjugated to CRM₁₉₇. In an embodiment of anyof the above second immunogenic compositions, the glycoconjugates fromS. pneumoniae serotypes 6A and 19A are conjugated to CRM₁₉₇. In anembodiment of any of the above second immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 3 is conjugated to CRM₁₉₇.In an embodiment of any of the above second immunogenic compositions,the glycoconjugates from S. pneumoniae serotype 22F is conjugated toCRM₁₉₇. In an embodiment of any of the above second immunogeniccompositions, the glycoconjugates from S. pneumoniae serotype 33F isconjugated to CRM₁₉₇.

In an embodiment, the glycoconjugates of any of the above secondimmunogenic compositions are all individually conjugated to CRM₁₉₇.

In an embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 4,5, 6B, 7F, 9V, 14 and/or 23F of any of the above second immunogeniccompositions are individually conjugated to PD.

In an embodiment, the glycoconjugate from S. pneumoniae serotype 18C ofany of the above second immunogenic compositions is conjugated to TT.

In an embodiment, the glycoconjugate from S. pneumoniae serotype 19F ofany of the above second immunogenic compositions is conjugated to DT.

In an embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 4,5, 6B, 7F, 9V, 14 and/or 23F of any of the above second immunogeniccompositions are individually conjugated to PD, the glycoconjugate fromS. pneumoniae serotype 18C is conjugated to TT and the glycoconjugatefrom S. pneumoniae serotype 19F is conjugated to DT.

In an embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 4,5, 6B, 7F, 9V, 14 and/or 23F of any of the above second immunogeniccompositions are individually conjugated to PD, the glycoconjugate fromS. pneumoniae serotype 18C is conjugated to TT, the glycoconjugate fromS. pneumoniae serotype 19F is conjugated to DT, the glycoconjugate fromS. pneumoniae serotype 22F is conjugated to CRM₁₉₇ and theglycoconjugate from S. pneumoniae serotype 33F is conjugated to CRM₁₉₇.

In an embodiment the above second immunogenic compositions comprise from7 to 15 different serotypes of S. pneumoniae. In one embodiment theabove second immunogenic compositions comprise glycoconjugates from 7,8, 9, 10, 11, 12, 13, 14 or 15 different serotypes. In one embodimentthe above second immunogenic compositions comprise glycoconjugates from10 to 15 different serotypes. In an embodiment the above secondimmunogenic composition is a 7, 8, 9, 10, 11, 12, 13, 14 or 15-valentpneumococcal conjugate composition. In an embodiment the above secondimmunogenic composition is a 10-valent pneumococcal conjugatecomposition. In an embodiment the above second immunogenic compositionis an 11-valent pneumococcal conjugate composition. In an embodiment theabove second immunogenic composition is a 12-valent pneumococcalconjugate composition. In an embodiment the above second immunogeniccomposition is a 13-valent pneumococcal conjugate composition. In anembodiment the above second immunogenic composition is a 14-valentpneumococcal conjugate composition. In an embodiment the above secondimmunogenic composition is a 15-valent pneumococcal conjugatecomposition. In an embodiment, the above second immunogenic compositionis a 7-valent pneumococcal conjugate composition wherein said 7conjugates consists of 7 glycoconjugates from S. pneumoniae serotypes 4,6B, 9V, 14, 18C, 19F and 23F individually conjugated to CRM₁₉₇.

In an embodiment, the above second immunogenic composition is a10-valent pneumococcal conjugate composition wherein said 10 conjugatesconsists of glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B,7F, 9V, 14 and 23F individually conjugated to PD, glycoconjugate from S.pneumoniae serotype 18C conjugated to TT and glycoconjugate from S.pneumoniae serotype 19F conjugated to DT.

In an embodiment, the above second immunogenic composition is an11-valent pneumococcal conjugate composition wherein said 11 conjugatesconsists of glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B,7F, 9V, 14 and 23F individually conjugated to PD, glycoconjugate from S.pneumoniae serotype 18C conjugated to TT, glycoconjugate from S.pneumoniae serotype 19F conjugated to DT and glycoconjugate from S.pneumoniae serotype 22F conjugated to CRM₁₉₇.

In an embodiment, the above second immunogenic composition is an11-valent pneumococcal conjugate composition wherein said 11 conjugatesconsists of glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B,7F, 9V, 14 and 23F individually conjugated to PD, glycoconjugate from S.pneumoniae serotype 18C conjugated to TT, glycoconjugate from S.pneumoniae serotype 19F conjugated to DT and glycoconjugate from S.pneumoniae serotype 33F conjugated to CRM₁₉₇.

In an embodiment, the above second immunogenic composition is a12-valent pneumococcal conjugate composition wherein said 12 conjugatesconsists of glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B,7F, 9V, 14 and 23F individually conjugated to PD, glycoconjugate from S.pneumoniae serotype 18C conjugated to TT, glycoconjugate from S.pneumoniae serotype 19F conjugated to DT, glycoconjugate from S.pneumoniae serotype 22F conjugated to CRM₁₉₇ and glycoconjugate from S.pneumoniae serotype 33F conjugated to CRM₁₉₇.

In an embodiment, the above second immunogenic composition is a13-valent pneumococcal conjugate composition wherein said 13 conjugatesconsists of glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19A, 19F and 23F individually conjugated to CRM₁₉₇.

In an embodiment, the above second immunogenic composition is a14-valent pneumococcal conjugate composition wherein said 14 conjugatesconsists of glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 22F individually conjugated toCRM₁₉₇.

In an embodiment, the above second immunogenic composition is a14-valent pneumococcal conjugate composition wherein said 14 conjugatesconsists of glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 33F individually conjugated toCRM₁₉₇.

In an embodiment, the above second immunogenic composition is a15-valent pneumococcal conjugate composition wherein said 15 conjugatesconsists of glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 22F and 33F individually conjugatedto CRM₁₉₇.

In an embodiment the dosage of the above second immunogenic is asdisclosed at section 5 below.

In an embodiment the above second immunogenic compositions furthercomprise antigen(s) from other pathogen(s), particularly from bacteriaand/or viruses such as disclosed at section 6 below.

In an embodiment the above second immunogenic compositions furthercomprise one or more adjuvants as disclosed at section 7 below.

In an embodiment the above second immunogenic compositions areformulated as disclosed at section 8 below.

In an embodiment, the immunogenic compositions of the invention (such asany of the ones of section 2 above) are used in combination withPREVNAR® (PREVENAR® in some countries) (heptavalent vaccine), SYNFLORIX®(a decavalent vaccine) and/or PREVNAR 13® (PREVENAR 13® in somecountries) (tridecavalent vaccine).

4. KIT OF THE PRESENT INVENTION

In an aspect, the invention provides a kit comprising: (a) a firstimmunogenic composition, as defined at section 2 above; and (b) a secondimmunogenic composition comprising at least one glycoconjugate from aStreptococcus pneumoniae serotype selected from serotypes 1, 3, 4, 5,6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 22F and 33F.

In an aspect, the invention provides a kit comprising: (a) a firstimmunogenic composition, as defined at section 2 above; and (b) a secondimmunogenic composition comprising at least one glycoconjugate from aStreptococcus pneumoniae serotype selected from serotypes 1, 3, 4, 5,6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

In an aspect, the invention provides a kit comprising: (a) a firstimmunogenic composition, as defined at section 2 above; and (b) a secondimmunogenic composition as defined at section 3 above.

1. In an embodiment the second immunogenic composition of the kit (part(b) of the kit) comprises glycoconjugates from S. pneumoniae serotypes4, 6B, 9V, 14, 18C, 19F and 23F (such as the glycoconjugates of section1.3.1 above).2. In another embodiment said second immunogenic composition comprisesin addition to point 1 above, at least one glycoconjugate from S.pneumoniae serotypes 1, 5 and 7F (such as the glycoconjugates of section1.3.1 above).3. In another embodiment said second immunogenic composition comprisesin addition to point 1 or 2 above, at least one glycoconjugate from S.pneumoniae serotypes 6A and 19A (such as the glycoconjugates of section1.3.1 above).4. In another embodiment said second immunogenic composition comprisesin addition to point 1, 2 or 3 above, at least one glycoconjugate fromS. pneumoniae serotype 3 (such as the glycoconjugates of section 1.3.1above).5. In another embodiment said second immunogenic composition comprisesin addition to point 1, 2, 3 or 4 above, at least one glycoconjugatefrom S. pneumoniae serotype 22F, such as the ones disclosed at section1.3.2 above.6. In another embodiment said second immunogenic composition comprisesin addition to point 1, 2, 3, 4 or 5 above, at least one glycoconjugatefrom S. pneumoniae serotype 33F such as the ones disclosed at section1.3.3 above.

In an embodiment the second immunogenic composition of the kit (part (b)of the kit) comprises glycoconjugates from S. pneumoniae serotypes 4,6B, 9V, 14, 18C, 19F and 23F (such as the glycoconjugates of section1.3.1 above).

In an embodiment the second immunogenic composition of the kit comprisesglycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14,18C, 19F and 23F (such as the glycoconjugates of section 1.3.1 above).

In an embodiment the second immunogenic composition of the kit comprisesglycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V,14, 18C, 19A, 19F and 23F (such as the glycoconjugates of section 1.3.1above).

In an embodiment the second immunogenic composition of the kit comprisesglycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V,14, 18C, 19A, 19F and 23F (such as the glycoconjugates of section 1.3.1above).

In an embodiment the second immunogenic composition of the kit comprisesglycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14,18C, 19F, 23F and 22F (such as the glycoconjugates of section 1.3.1 and1.3.2 above).

In an embodiment the second immunogenic composition of the kit comprisesglycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14,18C, 19F, 23F and 33F (such as the glycoconjugates of sections 1.3.1 and1.3.3 above).

In an embodiment the second immunogenic composition of the kit comprisesglycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14,18C, 19F, 23F, 22F and 33F (such as the glycoconjugates of section1.3.1, 1.3.2 and 1.3.3 above).

In an embodiment the second immunogenic composition of the kit comprisesglycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V,14, 18C, 19A, 19F, 23F and 22F (such as the glycoconjugates of sections1.3.1 and 1.3.2 above).

In an embodiment the second immunogenic composition of the kit comprisesglycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V,14, 18C, 19A, 19F, 23F and 33F (such as the glycoconjugates of sections1.3.1 and 1.3.3 above).

In an embodiment the second immunogenic composition of the kit comprisesglycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V,14, 18C, 19A, 19F, 23F, 22F and 33F (such as the glycoconjugates ofsections 1.3.1, 1.3.2 and 1.3.3 above).

Preferably, all the glycoconjugates of the second immunogeniccomposition of the kit are individually conjugated to the carrierprotein.

In an embodiment of any of the above kits, the glycoconjugates from S.pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F are conjugated toCRM₁₉₇. In an embodiment of any of the above kits, the glycoconjugatesfrom S. pneumoniae serotypes 1, 5 and 7F are conjugated to CRM₁₉₇. In anembodiment of any of the above kits, the glycoconjugates from S.pneumoniae serotypes 6A and 19A are conjugated to CRM₁₉₇. In anembodiment of any of the above kits, the glycoconjugates from S.pneumoniae serotype 3 is conjugated to CRM₁₉₇.

In an embodiment, the glycoconjugates of any of the above kits are allindividually conjugated to CRM₁₉₇.

In another embodiment, the glycoconjugates from S. pneumoniae serotypes1, 4, 5, 6B, 7F, 9V, 14 and/or 23F of any of the above kits areindividually conjugated to PD.

In an embodiment, the glycoconjugate from S. pneumoniae serotype 18C ofany of the above kits is conjugated to TT.

In an embodiment, the glycoconjugate from S. pneumoniae serotype 19F ofany of the above kits is conjugated to DT.

In an embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 4,5, 6B, 7F, 9V, 14 and/or 23F of any of the above kits are individuallyconjugated to PD, the glycoconjugate from S. pneumoniae serotype 18C isconjugated to TT and the glycoconjugate from S. pneumoniae serotype 19Fis conjugated to DT.

In an embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 4,5, 6B, 7F, 9V, 14 and/or 23F of any of the above kits are individuallyconjugated to PD, the glycoconjugate from S. pneumoniae serotype 18C isconjugated to TT, the glycoconjugate from S. pneumoniae serotype 19F isconjugated to DT, the glycoconjugate from S. pneumoniae serotype 22F isconjugated to CRM₁₉₇ and the glycoconjugate from S. pneumoniae serotype33F is conjugated to CRM₁₉₇.

In an embodiment the above second immunogenic compositions comprise from7 to 15 different serotypes of S. pneumoniae. In one embodiment theabove second immunogenic compositions comprise glycoconjugates from 7,8, 9, 10, 11, 12, 13, 14 or 15 different serotypes. In one embodimentthe above second immunogenic compositions comprise glycoconjugates from10 to 15 different serotypes. In an embodiment the above secondimmunogenic composition is a 7, 8, 9, 10, 11, 12, 13, 14 or 15-valentpneumococcal conjugate composition. In an embodiment the above secondimmunogenic composition is a 10-valent pneumococcal conjugatecomposition. In an embodiment the above second immunogenic compositionis an 11-valent pneumococcal conjugate composition. In an embodiment theabove second immunogenic composition is a 12-valent pneumococcalconjugate composition. In an embodiment the above second immunogeniccomposition is a 13-valent pneumococcal conjugate composition. In anembodiment the above second immunogenic composition is a 14-valentpneumococcal conjugate composition. In an embodiment the above secondimmunogenic composition is a 15-valent pneumococcal conjugatecomposition.

In an embodiment, the above second immunogenic composition is a 7-valentpneumococcal conjugate composition wherein said 7 conjugates consists of7 glycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19Fand 23F individually conjugated to CRM₁₉₇.

In an embodiment, the above second immunogenic composition is a10-valent pneumococcal conjugate composition wherein said 10 conjugatesconsists of glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B,7F, 9V, 14 and 23F individually conjugated to PD, glycoconjugate from S.pneumoniae serotype 18C conjugated to TT and glycoconjugate from S.pneumoniae serotype 19F conjugated to DT.

In an embodiment, the above second immunogenic composition is an11-valent pneumococcal conjugate composition wherein said 11 conjugatesconsists of glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B,7F, 9V, 14 and 23F individually conjugated to PD, glycoconjugate from S.pneumoniae serotype 18C conjugated to TT, glycoconjugate from S.pneumoniae serotype 19F conjugated to DT and glycoconjugate from S.pneumoniae serotype 22F conjugated to CRM₁₉₇.

In an embodiment, the above second immunogenic composition is an11-valent pneumococcal conjugate composition wherein said 11 conjugatesconsists of glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B,7F, 9V, 14 and 23F individually conjugated to PD, glycoconjugate from S.pneumoniae serotype 18C conjugated to TT, glycoconjugate from S.pneumoniae serotype 19F conjugated to DT and glycoconjugate from S.pneumoniae serotype 33F conjugated to CRM₁₉₇.

In an embodiment, the above second immunogenic composition is a12-valent pneumococcal conjugate composition wherein said 12 conjugatesconsists of glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B,7F, 9V, 14 and 23F individually conjugated to PD, glycoconjugate from S.pneumoniae serotype 18C conjugated to TT, glycoconjugate from S.pneumoniae serotype 19F conjugated to DT, glycoconjugate from S.pneumoniae serotype 22F conjugated to CRM₁₉₇ and glycoconjugate from S.pneumoniae serotype 33F conjugated to CRM₁₉₇.

In an embodiment, the above second immunogenic composition is a13-valent pneumococcal conjugate composition wherein said 13 conjugatesconsists of glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19A, 19F and 23F individually conjugated to CRM₁₉₇.

In an embodiment, the above second immunogenic composition is a14-valent pneumococcal conjugate composition wherein said 14 conjugatesconsists of glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 22F individually conjugated toCRM₁₉₇.

In an embodiment, the above second immunogenic composition is a14-valent pneumococcal conjugate composition wherein said 14 conjugatesconsists of glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 33F individually conjugated toCRM₁₉₇.

In an embodiment, the above second immunogenic composition is a15-valent pneumococcal conjugate composition wherein said 15 conjugatesconsists of glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 22F and 33F individually conjugatedto CRM₁₉₇.

In an embodiment the dosage of above second immunogenic is as disclosedat section 5 below.

In an embodiment the above second immunogenic compositions furthercomprise antigens from other pathogens, particularly from bacteriaand/or viruses such as disclosed at section 6 below.

In an embodiment the above second immunogenic compositions furthercomprise one or more adjuvants as disclosed at section 7 below.

In an embodiment the above second immunogenic compositions areformulated as disclosed at section 8 below.

In an embodiment, the immunogenic compositions of the invention (such asany of the ones of section 2 above) are used in combination withPREVNAR® (PREVENAR® in some countries) (heptavalent vaccine), SYNFLORIX®(a decavalent vaccine) and/or PREVNAR 13® (PREVENAR 13® in somecountries) (tridecavalent vaccine).

In an aspect of the present invention, the kit takes the form of twocontainers. Therefore, in one embodiment of the present invention eachof the immunogenic compositions of the kit (i.e., the first immunogeniccomposition and the second immunogenic composition) is comprised in aseparate container.

In one embodiment, the first immunogenic composition of the kit (part(a) of the kit) is comprised in a container selected from the groupconsisting of a vial, a syringe, a flask, a fermentor, a bioreactor, abag, a jar, an ampoule, a cartridge and a disposable pen. In certainembodiments, the container is siliconized.

In one embodiment, the second immunogenic composition of the kit (part(b) of the kit) is comprised in a container selected from the groupconsisting of a vial, a syringe, a flask, a fermentor, a bioreactor, abag, a jar, an ampoule, a cartridge and a disposable pen. In certainembodiments, the container is siliconized.

In an embodiment, the container is made of glass, metals (e.g., steel,stainless steel, aluminum, etc.) and/or polymers (e.g., thermoplastics,elastomers, thermoplastic-elastomers). In an embodiment, the containeris made of glass.

In one embodiment, the first and second immunogenic compositions of thekit are comprised in a syringe or a disposable pen. In one embodiment,the first and second immunogenic compositions of the kit are comprisedin a syringe. In certain embodiments, the syringes are siliconized. Incertain embodiments, the siliconized syringes are made of glass.

In an embodiment, the first and second immunogenic compositions of thekit are mixed extemporaneously for simultaneous administration.

In an embodiment, the first and second immunogenic compositions are inliquid form, preferably contained in two containers. In one embodiment,the first and second containers are separate chambers in a dual-chambersyringe such that, when actuated, liquid in the first container isintroduced into the second container. The resulting mixture can thenexit the syringe. The two immunogenic compositions are kept separateuntil ready for mixing.

In an embodiment, the first and/or second immunogenic composition of thekit is/are in lyophilized form.

In an embodiment, the first immunogenic composition of the kit is inlyophilized form and the second immunogenic composition is in liquidform. In another embodiment, the second immunogenic composition of thekit is in lyophilized form and the first immunogenic composition is inliquid form. In said embodiments, the lyophilised immunogeniccomposition can be reconstituted extemporaneously with the liquidimmunogenic composition for simultaneous administration of bothimmunogenic compositions.

In said embodiments, the kit contains two containers, one containerincludes liquid material for reconstitution and the second containerincludes lyophilised material. In one embodiment the second container ishermetically sealed. In an embodiment, the liquid material is introducedinto the second container via a first needle, thereby reconstituting thelyophilised material into a liquid form. The resulting mixture is thenwithdrawn, into a container (such as a syringe), for administration to apatient. In one embodiment the withdrawal step is via the first needle.In another embodiment, the withdrawal step is via a second needle. In anembodiment, the needle used for the withdrawal step is the same needlethat is used for patient injection. In another embodiment, the needleused for the withdrawal step is different from the needle used forpatient injection.

In one embodiment, the second container is a vial. In a furtherembodiment the first and second containers are separate chambers in adual-chamber syringe such that, when actuated, the liquid material isintroduced from the first container into the second container. Theresulting mixture exits the syringe in liquid form. In a preferredembodiment, the lyophilised and liquid materials are kept separate untilready for mixing.

In an embodiment, the kit comprises a ready-filled syringe and a vial.In one embodiment the syringe comprises a single dose of the firstimmongenic composition and the vial comprises a single dose of thesecond immunogenic composition. In an embodiment the syringe comprises asingle dose of the second immongenic composition and the vial comprisesa single dose of the first immunogenic composition. In anotherembodiment, the syringe and the vial comprise multiple doses.

5. DOSAGE OF THE IMMUNOGENIC COMPOSITIONS

The amount of glycoconjugate(s) in each dose is selected as an amountwhich induces an immunoprotective response without significant, adverseside effects in typical vaccinees. Such amount will vary depending uponwhich specific immunogen is employed and how it is presented.

5.1 Glycoconjugate Amount

The amount of a particular glycoconjugate in an immunogenic compositioncan be calculated based on total polysaccharide for that conjugate(conjugated and non-conjugated). For example, a glycoconjugate with 20%free polysaccharide has about 80 μg of conjugated polysaccharide andabout 20 μg of nonconjugated polysaccharide in a 100 μg polysaccharidedose. The amount of glycoconjugate can vary depending upon thepneumococcal serotype. The saccharide concentration can be determined bythe uronic acid assay.

The “immunogenic amount” of the different polysaccharide components inthe immunogenic composition, may diverge and each may comprise about 1μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7μg, about 8 μg, about 9 μg, about 10 μg, about 15 μg, about 20 μg, about30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg,about 90 μg, or about 100 μg of any particular polysaccharide antigen.

Generally, each dose comprises 0.1 μg to 100 μg of polysaccharide for agiven serotype, particularly 0.5 μg to 20 μg, more particularly 1.0 μgto 10 μg, and even more more particularly 2.0 μg to 5.0 μg. Any wholenumber integer within any of the above ranges is contemplated as anembodiment of the disclosure.

In an embodiment, each dose comprises about 1.0 μg, about 1.2 μg, about1.4 μg, about 1.6 μg, about 1.8 μg, about 2.0 μg, about 2.2 μg, about2.4 μg, about 2.6 μg, about 2.8 μg, about 3.0 μg, about 3.2 μg, about3.4 μg, about 3.6 μg, about 3.8 μg, about 4.0 μg, about 4.2 μg, about4.4 μg, about 4.6 μg, about 4.8 μg, about 5.0 μg, about 5.2 μg, about5.4 μg, about 5.6 μg, about 5.8 μg or about 6.0 μg of polysaccharide foreach particular glycoconjugate.

In an embodiment, each dose comprises about 1.1 μg, about 1.2 μg, about1.3 μg, about 1.4 μg, about 1.5 μg, about 1.6 μg, about 1.7 μg, about1.8 μg, about 1.9 μg, about 2.0 μg, about 2.1 μg, about 2.2 μg, about2.3 μg, about 2.4 μg, about 2.5 μg, about 2.6 μg, about 2.7 μg, about2.8 μg, about 2.9 μg, or about 3.0 μg μg of polysaccharide forglycoconjugates from S. pneumoniae serotype 1, 3, 4, 5, 6A, 7F, 8, 9V,10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and/or 33F.

In an embodiment, each dose will comprise about 1.1 μg, about 1.2 μg,about 1.3 μg, about 1.4 μg, about 1.5 μg, about 1.6 μg, about 1.7 μg,about 1.8 μg, about 1.9 μg, about 2.0 μg, about 2.1 μg, about 2.2 μg,about 2.3 μg, about 2.4 μg, about 2.5 μg, about 2.6 μg, about 2.7 μg,about 2.8 μg, about 2.9 μg, or about 3.0 μg μg of polysaccharide forglycoconjugates from S. pneumoniae serotype 8, 10A, 11A, 12F, 15B, 22Fand 33F.

In an embodiment, each dose comprises about 2.0 μg, about 2.2 μg, about2.4 μg, about 2.6 μg, about 2.8 μg, about 3.0 μg, about 3.2 μg, about3.4 μg, about 3.6 μg, about 3.8 μg, about 4.0 μg, about 4.2 μg, about4.4 μg, about 4.6 μg, about 4.8 μg, about 5.0, about 5.2 μg, about 5.4μg, about 5.6 μg, about 5.8 μg or about 6.0 μg of polysaccharide forglycoconjugates from S. pneumoniae serotype 6B.

In an embodiment, each dose comprise about 1.5 μg to about 3.0 μg ofpolysaccharide for each glycoconjugate from S. pneumoniae serotype 1, 3,4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23Fand/or 33F, and about 3.0 μg to about 6.0 μg of polysaccharide forglycoconjugate from S. pneumoniae serotype 6B.

In an embodiment, each dose comprises about 2.0 μg to about 2.5 μg ofpolysaccharide for each glycoconjugate from S. pneumoniae serotype 1, 3,4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23Fand/or 33F, and about 4.0 μg to about 4.8 μg of polysaccharide forglycoconjugate from S. pneumoniae serotype 6B.

In an embodiment, each dose comprises about 2.2 μg of polysaccharidefrom each glycoconjugate from S. pneumoniae serotype 1, 3, 4, 5, 6A, 7F,8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and/or 33F, andabout 4.4 μg of polysaccharide for glycoconjugate from S. pneumoniaeserotype 6B.

In an embodiment, each dose comprises about 1.5 μg to about 3.0 μg ofpolysaccharide for each glycoconjugate from S. pneumoniae serotype 8,10A, 11A, 12F, 15B, 22F and 33F

In an embodiment, each dose comprises about 2.0 μg to about 2.5 μg ofpolysaccharide for each glycoconjugate from S. pneumoniae serotype 8,10A, 11A, 12F, 15B, 22F and 33F.

In an embodiment, each dose comprises about 2.2 μg of polysaccharidefrom each glycoconjugate from S. pneumoniae serotype 8, 10A, 11A, 12F,15B, 22F and 33F.

5.2 Carrier Amount

Generally, each dose of an immunogenic composition of the inventioncomprises 1 μg to 150 μg of carrier protein, particularly 10 μg to 100μg of carrier protein, more particularly 15 μg to 50 μg of carrierprotein, and even more particularly 16 μg to 40 μg of carrier protein.In an embodiment, said carrier protein is CRM₁₉₇.

In an embodiment, each dose comprises about 1 μg, about 2 μg, about 3μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg,about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg,about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg,about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg,about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg,about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about64 μg, about 65 μg, about 66 μg, about 67 μg, about 68 μg, about 69 μg,about 70 μg, about 71 μg, about 72 μg, about 73 μg, about 74 μg or about75 μg of carrier protein. In an embodiment, said carrier protein isCRM₁₉₇.

In an embodiment, each dose comprises about about 10 μg, about 11 μg,about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg,about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about28 μg, about 29 μg, or about 30 μg of carrier protein. In an embodiment,said carrier protein is CRM₁₉₇.

6. FURTHER ANTIGENS

Immunogenic compositions disclosed herein comprise conjugated S.pneumoniae saccharide antigen(s) (glycoconjugate(s)). They may alsofurther include at least one antigen from other pathogens, particularlyfrom bacteria and/or viruses.

In an embodiment, the immunogenic composition disclosed herein furthercomprises at least one antigen selected from the group consisting of adiphtheria toxoid (D), a tetanus toxoid (T), a pertussis antigen (P), anacellular pertussis antigen (Pa), a hepatitis B virus (HBV) surfaceantigen (HBsAg), a hepatitis A virus (HAV) antigen, a conjugatedHaemophilus influenzae type b capsular saccharide (Hib), and inactivatedpoliovirus vaccine (IPV).

In an embodiment, the immunogenic compositions disclosed herein compriseD-T-Pa. In an embodiment, the immunogenic compositions disclosed hereincomprise D-T-Pa-Hib, D-T-Pa-IPV or D-T-Pa-HBsAg. In an embodiment, theimmunogenic compositions disclosed herein comprise D-T-Pa-HBsAg-IPV orD-T-Pa-HBsAg-Hib. In an embodiment, the immunogenic compositionsdisclosed herein comprise D-T-Pa-HBsAg-IPV-Hib.

Pertussis antigens: Bordetella pertussis causes whooping cough.Pertussis antigens in vaccines are either cellular (whole cell, in theform of inactivated B. pertussis cells) or acellular. Preparation ofcellular pertussis antigens is well documented (e.g., it may be obtainedby heat inactivation of phase I culture of B. pertussis). Preferably,however, the invention uses acellular antigens. Where acellular antigensare used, it is preferred to use one, two or (preferably) three of thefollowing antigens: (1) detoxified pertussis toxin (pertussis toxoid, orPT); (2) filamentous hemagglutinin (FHA); (3) pertactin (also known asthe 69 kiloDalton outer membrane protein). FHA and pertactin may betreated with formaldehyde prior to use according to the invention. PT ispreferably detoxified by treatment with formaldehyde and/orglutaraldehyde. Acellular pertussis antigens are preferably adsorbedonto one or more aluminum salt adjuvants. As an alternative, they may beadded in an unadsorbed state. Where pertactin is added, it is preferablyalready adsorbed onto an aluminum hydroxide adjuvant. PT and FHA may beadsorbed onto an aluminum hydroxide adjuvant or an aluminum phosphate.Adsorption of all of PT, FHA and pertactin to aluminum hydroxide is mostpreferred.

Inactivated poliovirus vaccine: Poliovirus causes poliomyelitis. Ratherthan use oral poliovirus vaccine, preferred embodiments of the inventionuse IPV. Prior to administration to patients, polioviruses must beinactivated, and this can be achieved by treatment with formaldehyde.Poliomyelitis can be caused by one of three types of poliovirus. Thethree types are similar and cause identical symptoms, but they areantigenically different and infection by one type does not protectagainst infection by others. It is therefore preferred to use threepoliovirus antigens in the invention: poliovirus Type 1 (e.g., Mahoneystrain), poliovirus Type 2 (e.g., MEF-1 strain), and poliovirus Type 3(e.g., Saukett strain). The viruses are preferably grown, purified andinactivated individually, and are then combined to give a bulk trivalentmixture for use with the invention.

Diphtheria toxoid: Corynebacterium diphtheriae causes diphtheria.Diphtheria toxin can be treated (e.g., using formalin or formaldehyde)to remove toxicity while retaining the ability to induce specificanti-toxin antibodies after injection. These diphtheria toxoids are usedin diphtheria vaccines. Preferred diphtheria toxoids are those preparedby formaldehyde treatment. The diphtheria toxoid can be obtained bygrowing C. diphtheriae in growth medium, followed by formaldehydetreatment, ultrafiltration and precipitation. The toxoided material maythen be treated by a process comprising sterile filtration and/ordialysis. The diphtheria toxoid is preferably adsorbed onto an aluminumhydroxide adjuvant.

Tetanus toxoid: Clostridium tetani causes tetanus. Tetanus toxin can betreated to give a protective toxoid. The toxoids are used in tetanusvaccines. Preferred tetanus toxoids are those prepared by formaldehydetreatment. The tetanus toxoid can be obtained by growing C. tetani ingrowth medium, followed by formaldehyde treatment, ultrafiltration andprecipitation. The material may then be treated by a process comprisingsterile filtration and/or dialysis.

Hepatitis A virus antigens: Hepatitis A virus (HAV) is one of the knownagents which causes viral hepatitis. A preferred HAV component is basedon inactivated virus, and inactivation can be achieved by formalintreatment.

Hepatitis B virus (HBV) is one of the known agents which causes viralhepatitis. The major component of the capsid is a protein known as HBVsurface antigen or, more commonly, HBsAg, which is typically a 226-aminoacid polypeptide with a molecular weight of ˜24 kDa. All existinghepatitis B vaccines contain HBsAg, and when this antigen isadministered to a normal vaccinee, it stimulates the production ofanti-HBsAg antibodies which protect against HBV infection.

For vaccine manufacture, HBsAg has been made in two ways: purificationof the antigen in particulate form from the plasma of chronic hepatitisB carriers or expression of the protein by recombinant DNA methods(e.g., recombinant expression in yeast cells). Unlike native HBsAg(i.e., as in the plasma-purified product), yeast-expressed HBsAg isgenerally non-glycosylated, and this is the most preferred form of HBsAgfor use with the invention.

Conjugated Haemophilus influenzae type b antigens: Haemophilusinfluenzae type b (Hib) causes bacterial meningitis. Hib vaccines aretypically based on the capsular saccharide antigen, the preparation ofwhich is well documented. The Hib saccharide can be conjugated to acarrier protein in order to enhance its immunogenicity, especially inchildren. Typical carrier proteins are tetanus toxoid, diphtheriatoxoid, CRM₁₉₇ , H. influenzae protein D, and an outer membrane proteincomplex from serogroup B meningococcus. The saccharide moiety of theconjugate may comprise full-length polyribosylribitol phosphate (PRP) asprepared from Hib bacteria, and/or fragments of full-length PRP. Hibconjugates may or may not be adsorbed to an aluminum salt adjuvant.

In an embodiment the immunogenic compositions disclosed herein furtherinclude a conjugated N. meningitidis serogroup Y capsular saccharide(MenY), and/or a conjugated N. meningitidis serogroup C capsularsaccharide (MenC).

In an embodiment the immunogenic compositions disclosed herein furtherinclude a conjugated N. meningitidis serogroup A capsular saccharide(MenA), a conjugated N. meningitidis serogroup W135 capsular saccharide(MenW135), a conjugated N. meningitidis serogroup Y capsular saccharide(MenY), and/or a conjugated N. meningitidis serogroup C capsularsaccharide (MenC).

In an embodiment the immunogenic compositions disclosed herein furtherinclude a conjugated N. meningitidis serogroup W135 capsular saccharide(MenW135), a conjugated N. meningitidis serogroup Y capsular saccharide(MenY), and/or a conjugated N. meningitidis serogroup C capsularsaccharide (MenC).

An aspect of the invention provides a kit as defined at section 4 abovewherein any of the above further antigen(s) is part of the firstimmunogenic composition (part (a) of the kit).

An aspect of the invention provides a kit as defined at section 4 abovewherein any of the above further antigen(s) is part of the secondimmunogenic composition (part (b) of the kit).

An aspect of the invention provides a kit as defined at section 4 abovewherein any of the above further antigen(s) is part of the firstimmunogenic composition (part (a) of the kit) and any of the abovefurther antigen(s) is part of the second immunogenic composition (part(b) of the kit).

7. ADJUVANT(S)

In some embodiments, the immunogenic compositions disclosed herein mayfurther comprise at least one, two or three adjuvants. The term“adjuvant” refers to a compound or mixture that enhances the immuneresponse to an antigen. Antigens may act primarily as a delivery system,primarily as an immune modulator or have strong features of both.Suitable adjuvants include those suitable for use in mammals, includinghumans.

Examples of known suitable delivery-system type adjuvants that can beused in humans include, but are not limited to, alum (e.g., aluminumphosphate, aluminum sulfate or aluminum hydroxide), calcium phosphate,liposomes, oil-in-water emulsions such as MF59 (4.3% w/v squalene, 0.5%w/v polysorbate 80 (TWEEN® 80), 0.5% w/v sorbitan trioleate (Span 85)),water-in-oil emulsions such as MONTANIDE™, andpoly(D,L-lactide-co-glycolide) (PLG) microparticles or nanoparticles.

In an embodiment, the immunogenic compositions disclosed herein comprisealuminum salts (alum) as adjuvant (e.g., aluminum phosphate, aluminumsulfate or aluminum hydroxide). In a preferred embodiment, theimmunogenic compositions disclosed herein comprise aluminum phosphate oraluminum hydroxide as adjuvant. In an embodiment, the immunogeniccompositions disclosed herein comprise from 0.1 mg/mL to 1 mg/mL or from0.2 mg/mL to 0.3 mg/mL of elemental aluminum in the form of aluminumphosphate. In an embodiment, the immunogenic compositions disclosedherein comprise about 0.25 mg/mL of elemental aluminum in the form ofaluminum phosphate. Examples of known suitable immune modulatory typeadjuvants that can be used in humans include, but are not limited to,saponin extracts from the bark of the Aquilla tree (QS21, QUILA®), TLR4agonists such as MPL (Monophosphoryl Lipid A), 3DMPL (3-O-deacylatedMPL) or GLA-AQ, LT/CT mutants, cytokines such as the variousinterleukins (e.g., IL-2, IL-12) or GM-CSF, and the like.

Examples of known suitable immune modulatory type adjuvants with bothdelivery and immune modulatory features that can be used in humansinclude, but are not limited to, ISCOMS (see, e.g., Sjölander et al.(1998) J. Leukocyte Biol. 64:713; WO 90/03184, WO 96/11711, WO 00/48630,WO 98/36772, WO 00/41720, WO 2006/134423 and WO 2007/026190) or GLA-EMwhich is a combination of a TLR4 agonist and an oil-in-water emulsion.

For veterinary applications including but not limited to animalexperimentation, one can use Complete Freund's Adjuvant (CFA), Freund'sIncomplete Adjuvant (IFA), EMULSIGEN®,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI™, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/TWEEN®80 emulsion.

Further exemplary adjuvants to enhance effectiveness of the pneumococcalvaccines as disclosed herein include, but are not limited to: (1)oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (see below) orbacterial cell wall components), such as for example (a) SAF, containing10% Squalane, 0.4% TWEEN® 80, 5% pluronic-blocked polymer L121, andthr-MDP either microfluidized into a submicron emulsion or vortexed togenerate a larger particle size emulsion, and (b) RIBI™ adjuvant system(RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2%TWEEN® 80, and one or more bacterial cell wall components such asmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (DETOX™); (2) saponin adjuvants, suchas QS21, STIMULON™ (Cambridge Bioscience, Worcester, Mass.), ABISCO®(Isconova, Sweden), or ISCOMATRIX® (Commonwealth Serum Laboratories,Australia), may be used or particles generated therefrom such as ISCOMs(immunostimulating complexes), which ISCOMS may be devoid of additionaldetergent (e.g., WO 00/07621); (3) Complete Freund's Adjuvant (CFA) andIncomplete Freund's Adjuvant (IFA); (4) cytokines, such as interleukins(e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (e.g., WO 99/44636)),interferons (e.g., gamma interferon), macrophage colony stimulatingfactor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryllipid A (MPL) or 3-O-deacylated MPL (3dMPL) (see, e.g., GB-2220221,EP0689454), optionally in the substantial absence of alum when used withpneumococcal saccharides (see, e.g., WO 00/56358); (6) combinations of3dMPL with, for example, QS21 and/or oil-in-water emulsions (see, e.g.,EP0835318, EP0735898, EP0761231); (7) a polyoxyethylene ether or apolyoxyethylene ester (see, e.g., WO 99/52549); (8) a polyoxyethylenesorbitan ester surfactant in combination with an octoxynol (e.g., WO01/21207) or a polyoxyethylene alkyl ether or ester surfactant incombination with at least one additional non-ionic surfactant such as anoctoxynol (e.g., WO 01/21152); (9) a saponin and an immunostimulatoryoligonucleotide (e.g., a CpG oligonucleotide) (e.g., WO 00/62800); (10)an immunostimulant and a particle of metal salt (see, e.g., WO00/23105); (11) a saponin and an oil-in-water emulsion (e.g., WO99/11241); (12) a saponin (e.g., QS21)+3dMPL+IM2 (optionally+a sterol)(e.g., WO 98/57659); (13) other substances that act as immunostimulatingagents to enhance the efficacy of the composition. Muramyl peptidesinclude N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), etc.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise a CpG Oligonucleotide as adjuvant. A CpGoligonucleotide as used herein refers to an immunostimulatory CpGoligodeoxynucleotide (CpG ODN), and accordingly these terms are usedinterchangeably unless otherwise indicated. Immunostimulatory CpGoligodeoxynucleotides contain one or more immunostimulatory CpG motifsthat are unmethylated cytosine-guanine dinucleotides, optionally withincertain preferred base contexts. The methylation status of the CpGimmunostimulatory motif generally refers to the cytosine residue in thedinucleotide. An immunostimulatory oligonucleotide containing at leastone unmethylated CpG dinucleotide is an oligonucleotide which contains a5′ unmethylated cytosine linked by a phosphate bond to a 3′ guanine, andwhich activates the immune system through binding to Toll-like receptor9 (TLR-9). In another embodiment the immunostimulatory oligonucleotidemay contain one or more methylated CpG dinucleotides, which willactivate the immune system through TLR9 but not as strongly as if theCpG motif(s) was/were unmethylated. CpG immunostimulatoryoligonucleotides may comprise one or more palindromes that in turn mayencompass the CpG dinucleotide. CpG oligonucleotides have been describedin a number of issued patents, published patent applications, and otherpublications, including U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806;6,218,371; 6,239,116; and 6,339,068.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise any of the CpG Oligonucleotide described atpage 3, line 22, to page 12, line 36, of WO 2010/125480.

Different classes of CpG immunostimulatory oligonucleotides have beenidentified. These are referred to as A, B, C and P class, and aredescribed in greater detail at page 3, line 22, to page 12, line 36, ofWO 2010/125480. Methods of the invention embrace the use of thesedifferent classes of CpG immunostimulatory oligonucleotides.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise an A class CpG oligonucleotide. Preferably,the “A class” CpG oligonucleotide of the invention has the followingnucleic acid sequence: 5′ GGGGACGACGTCGTGGGGGGG 3′ (SEQ ID NO: 1). Somenon-limiting examples of A-Class oligonucleotides include: 5′G*G*G_G_A_C_G_A_C_G_T_C_G_T_G_G*G*G*G*G*G 3′ (SEQ ID NO: 2); wherein “*”refers to a phosphorothioate bond and “_” refers to a phosphodiesterbond.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise a B class CpG Oligonucleotide. In oneembodiment, the CpG oligonucleotide for use in the present invention isa B class CpG oligonucleotide represented by at least the formula:

5′ X₁X₂CGX₃X₄ 3′, wherein X₁, X₂, X₃, and X₄ are nucleotides. In oneembodiment, X₂ is adenine, guanine, or thymine. In another embodiment,X₃ is cytosine, adenine, or thymine.

The B class CpG oligonucleotide sequences of the invention are thosebroadly described above as well as disclosed in WO 96/02555, WO 98/18810and U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116and 6,339,068. Exemplary sequences include but are not limited to thosedisclosed in these latter applications and patents.

In an embodiment, the “B class” CpG oligonucleotide of the invention hasthe following nucleic acid sequence:

(SEQ ID NO: 3) 5′ TCGTCGTTTTTCGGTGCTTTT 3′, or (SEQ ID NO: 4)5′ TCGTCGTTTTTCGGTCGTTTT 3′, or (SEQ ID NO: 5)5′ TCGTCGTTTTGTCGTTTTGTCGTT 3′, or (SEQ ID NO: 6)5′ TCGTCGTTTCGTCGTTTTGTCGTT 3′, or (SEQ ID NO: 7).5′ TCGTCGTTTTGTCGTTTTTTTCGA 3′.

In any of these sequences, all of the linkages may be allphosphorothioate bonds. In another embodiment, in any of thesesequences, one or more of the linkages may be phosphodiester, preferablybetween the “C” and the “G” of the CpG motif making a semi-soft CpGoligonucleotide. In any of these sequences, an ethyl-uridine or ahalogen may substitute for the 5′ T; examples of halogen substitutionsinclude but are not limited to bromo-uridine or iodo-uridinesubstitutions.

Some non-limiting examples of B-Class oligonucleotides include:

(SEQ ID NO: 8) 5′ T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*G*C*T*T*T*T 3′, or (SEQID NO: 9) 5′ T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T 3′, or (SEQ IDNO: 10) 5′ T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T* T 3′, or (SEQID NO: 11) 5′ T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*T*C*G*T* T 3′, or(SEQ ID NO: 12) 5′ T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*T*T*T*C*G* A 3′.wherein “*” refers to a phosphorothioate bond.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise a C class CpG Oligonucleotide. In anembodiment, the “C class” CpG oligonucleotides of the invention have thefollowing nucleic acid sequence:

(SEQ ID NO: 13) 5′ TCGCGTCGTTCGGCGCGCGCCG 3′, or (SEQ ID NO: 14)5′ TCGTCGACGTTCGGCGCGCGCCG 3′, or (SEQ ID NO: 15)5′ TCGGACGTTCGGCGCGCGCCG 3′, or (SEQ ID NO: 16) 5′ TCGGACGTTCGGCGCGCCG3′, or (SEQ ID NO: 17) 5′ TCGCGTCGTTCGGCGCGCCG 3′, or (SEQ ID NO: 18)5′ TCGACGTTCGGCGCGCGCCG 3′, or (SEQ ID NO: 19) 5′ TCGACGTTCGGCGCGCCG 3′,or (SEQ ID NO: 20) 5′ TCGCGTCGTTCGGCGCCG 3′, or (SEQ ID NO: 21)5′ TCGCGACGTTCGGCGCGCGCCG 3′, or (SEQ ID NO: 22)5′ TCGTCGTTTTCGGCGCGCGCCG 3′, or (SEQ ID NO: 23)5′ TCGTCGTTTTCGGCGGCCGCCG 3′, or (SEQ ID NO: 24)5′ TCGTCGTTTTACGGCGCCGTGCCG 3′, or (SEQ ID NO: 25)5′ TCGTCGTTTTCGGCGCGCGCCGT 3′.

In any of these sequences, all of the linkages may be allphosphorothioate bonds. In another embodiment, in any of thesesequences, one or more of the linkages may be phosphodiester, preferablybetween the “C” and the “G” of the CpG motif making a semi-soft CpGoligonucleotide.

Some non-limiting examples of C-Class oligonucleotides include:

(SEQ ID NO: 26) 5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′, or(SEQ ID NO: 27) 5′ T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C* G 3′, or(SEQ ID NO: 28) 5′ T*C_G*G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′, or (SEQID NO: 29) 5′ T*C_G*G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3′, or (SEQ ID NO:30) 5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3′, or (SEQ ID NO: 31)5′ T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′, or (SEQ ID NO: 32)5′ T*C_G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3′, or (SEQ ID NO: 33)5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*C*G 3′, or (SEQ ID NO: 34)5′ T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′, or (SEQ ID NO: 35)5′ T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G 3′, or (SEQ ID NO: 36)5′ T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*G*C*C*G*C*C*G 3′, or (SEQ ID NO: 37)5′ T*C*G*T*C_G*T*T*T*T*A*C_G*G*C*G*C*C_G*T*G*C*C* G 3′, or (SEQ ID NO:38) 5′ T*C_G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3′wherein “*” refers to a phosphorothioate bond and “_” refers to aphosphodiester bond. In any of these sequences, an ethyl-uridine or ahalogen may substitute for the 5′ T; examples of halogen substitutionsinclude but are not limited to bromo-uridine or iodo-uridinesubstitutions.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise a P class CpG Oligonucleotide. In anembodiment, the CpG oligonucleotide for use in the present invention isa P class CpG oligonucleotide containing a 5′ TLR activation domain andat least two palindromic regions, one palindromic region being a 5′palindromic region of at least 6 nucleotides in length and connected toa 3′ palindromic region of at least 8 nucleotides in length eitherdirectly or through a spacer, wherein the oligonucleotide includes atleast one YpR dinucleotide. In an embodiment, said oligonucleotide isnot T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G (SEQ ID NO: 27). Inone embodiment the P class CpG oligonucleotide includes at least oneunmethylated CpG dinucleotide. In another embodiment the TLR activationdomain is TCG, TTCG, TTTCG, TYpR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT,or TTTT. In yet another embodiment the TLR activation domain is withinthe 5′ palindromic region. In another embodiment the TLR activationdomain is immediately 5′ to the 5′ palindromic region.

In an embodiment, the “P class” CpG oligonucleotides of the inventionhave the following nucleic acid sequence: 5′ TCGTCGACGATCGGCGCGCGCCG 3′(SEQ ID NO: 39).

In said sequences, all of the linkages may be all phosphorothioatebonds. In another embodiment, one or more of the linkages may bephosphodiester, preferably between the “C” and the “G” of the CpG motifmaking a semi-soft CpG oligonucleotide. In any of these sequences, anethyl-uridine or a halogen may substitute for the 5′ T; examples ofhalogen substitutions include but are not limited to bromo-uridine oriodo-uridine substitutions.

A non-limiting example of P-Class oligonucleotides include:

(SEQ ID NO: 40) 5′ T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G 3′wherein “*” refers to a phosphorothioate bond and “_” refers to aphosphodiester bond. In one embodiment the oligonucleotide includes atleast one phosphorothioate linkage. In another embodiment allinternucleotide linkages of the oligonucleotide are phosphorothioatelinkages. In another embodiment the oligonucleotide includes at leastone phosphodiester-like linkage. In another embodiment thephosphodiester-like linkage is a phosphodiester linkage. In anotherembodiment a lipophilic group is conjugated to the oligonucleotide. Inone embodiment the lipophilic group is cholesterol.

In an embodiment, all the internucleotide linkages of the CpGoligonucleotides disclosed herein are phosphodiester bonds (“soft”oligonucleotides, as described in WO 2007/026190). In anotherembodiment, CpG oligonucleotides of the invention are rendered resistantto degradation (e.g., are stabilized). A “stabilized oligonucleotide”refers to an oligonucleotide that is relatively resistant to in vivodegradation (e.g., via an exo- or endo-nuclease). Nucleic acidstabilization can be accomplished via backbone modifications.Oligonucleotides having phosphorothioate linkages provide maximalactivity and protect the oligonucleotide from degradation byintracellular exo- and endo-nucleases.

The immunostimulatory oligonucleotides may have a chimeric backbone,which have combinations of phosphodiester and phosphorothioate linkages.For purposes of the instant invention, a chimeric backbone refers to apartially stabilized backbone, wherein at least one internucleotidelinkage is phosphodiester or phosphodiester-like, and wherein at leastone other internucleotide linkage is a stabilized internucleotidelinkage, wherein the at least one phosphodiester or phosphodiester-likelinkage and the at least one stabilized linkage are different. When thephosphodiester linkage is preferentially located within the CpG motifsuch molecules are called “semi-soft” as described in WO 2007/026190.

Other modified oligonucleotides include combinations of phosphodiester,phosphorothioate, methylphosphonate, methylphosphorothioate,phosphorodithioate, and/or p-ethoxy linkages.

Mixed backbone modified ODN may be synthesized as described in WO2007/026190. The size of the CpG oligonucleotide (i.e., the number ofnucleotide residues along the length of the oligonucleotide) also maycontribute to the stimulatory activity of the oligonucleotide. Forfacilitating uptake into cells, CpG oligonucleotide of the inventionpreferably have a minimum length of 6 nucleotide residues.Oligonucleotides of any size greater than 6 nucleotides (even many kblong) are capable of inducing an immune response if sufficientimmunostimulatory motifs are present, because larger oligonucleotidesare degraded inside cells. In certain embodiments, the CpGoligonucleotides are 6 to 100 nucleotides long, preferentially 8 to 30nucleotides long. In important embodiments, nucleic acids andoligonucleotides of the invention are not plasmids or expressionvectors.

In an embodiment, the CpG oligonucleotide disclosed herein comprisesubstitutions or modifications, such as in the bases and/or sugars asdescribed at paragraphs 134 to 147 of WO 2007/026190.

In an embodiment, the CpG oligonucleotide of the present invention ischemically modified. Examples of chemical modifications are known to theskilled person and are described, for example in Uhlmann et al. (1990)Chem. Rev. 90:543; S. Agrawal, Ed., Humana Press, Totowa, USA 1993;Crooke et al. (1996) Annu. Rev. Pharmacol. Toxicol. 36:107-129; andHunziker et al. (1995) Mod. Synth. Methods 7:331-417. An oligonucleotideaccording to the invention may have one or more modifications, whereineach modification is located at a particular phosphodiesterinternucleoside bridge and/or at a particular R-D-ribose unit and/or ata particular natural nucleoside base position in comparison to anoligonucleotide of the same sequence which is composed of natural DNA orRNA.

In some embodiments of the invention, CpG-containing nucleic acids mightbe simply mixed with immunogenic carriers according to methods known tothose skilled in the art (see, e.g., WO 03/024480).

In a particular embodiment of the present invention, any of theimmunogenic compositions disclosed herein comprise from 2 μg to 100 mgof CpG oligonucleotide, preferably from 0.1 mg to 50 mg CpGoligonucleotide, preferably from 0.2 mg to 10 mg CpG oligonucleotide,preferably from 0.3 mg to 5 mg CpG oligonucleotide, preferably from 0.3mg to 5 mg CpG oligonucleotide, even more preferably from 0.5 to 2 mgCpG oligonucleotide, even more preferably from 0.75 to 1.5 mg CpGoligonucleotide. In a preferred embodiment, any of the immunogeniccomposition disclosed herein comprises about 1 mg CpG oligonucleotide.

In an embodiment, the immunogenic composition of the invention (such asdefined at section 2 above), comprises an adjuvant as defined above,preferably an aluminum salt (alum) (e.g., aluminum phosphate, aluminumsulfate or aluminum hydroxide). In an embodiment, the immunogeniccomposition of the invention comprise aluminum phosphate or aluminumhydroxide as adjuvant.

In an embodiment, the immunogenic composition which may be used incombination with the immunogenic composition of the invention (such asdefined at section 3 above), comprises an adjuvant as defined above,preferably an aluminum salt (alum) (e.g., aluminum phosphate, aluminumsulfate or aluminum hydroxide). In an embodiment, said immunogeniccompositions comprise aluminum phosphate or aluminum hydroxide asadjuvant.

An aspect of the invention provides a kit as defined at section 4 abovewherein only the first immunogenic composition (part (a) of the kit)comprises an adjuvant as defined above.

An aspect of the invention provides a kit as defined at section 4 abovewherein only the second immunogenic composition (part (b) of the kit)comprises an adjuvant as defined above.

An aspect of the invention provides a kit as defined at section 4 abovewherein both immunogenic compositions (part (a) and (b) of the kit)comprise an adjuvant as defined above.

An aspect of the invention provides a kit as defined at section 4 abovewherein both immunogenic compositions (part (a) and (b) of the kit)comprise an adjuvant selected from the group consisting of aluminumphosphate, aluminum sulfate and aluminum hydroxide.

An aspect of the invention provides a kit as defined at section 4 abovewherein both immunogenic compositions (part (a) and (b) of the kit)comprise aluminum phosphate as adjuvant.

An aspect of the invention provides a kit as defined at section 4 abovewherein both immunogenic compositions (part (a) and (b) of the kit)comprise aluminium hydroxide as adjuvant.

An aspect of the invention provides a kit as defined at section 4 abovewherein both immunogenic compositions (part (a) and (b) of the kit)comprise aluminium sulfate as adjuvant.

8. FORMULATION

The immunogenic compositions disclosed herein may be formulated inliquid form (i.e., solutions or suspensions) or in a lyophilized form.Liquid formulations may advantageously be administered directly fromtheir packaged form and are thus ideal for injection without the needfor reconstitution in aqueous medium as otherwise required forlyophilized compositions.

Formulation of the immunogenic composition disclosed herein can beaccomplished using art-recognized methods. For instance, the individualpneumococcal conjugates can be formulated with a physiologicallyacceptable vehicle to prepare the composition. Examples of such vehiclesinclude, but are not limited to, water, buffered saline, polyols (e.g.,glycerol, propylene glycol, liquid polyethylene glycol) and dextrosesolutions.

The present disclosure provides an immunogenic composition comprisingany combination of glycoconjugates disclosed herein and apharmaceutically acceptable excipient, carrier, or diluent.

In an embodiment, the immunogenic composition disclosed herein is inliquid form, preferably in aqueous liquid form.

Immunogenic compositions of the disclosure may comprise one or more of abuffer, a salt, a divalent cation, a non-ionic detergent, acryoprotectant such as a sugar, and an anti-oxidant such as a freeradical scavenger or chelating agent, or any combinations thereof.

In an embodiment, the immunogenic compositions disclosed herein comprisea buffer. In an embodiment, said buffer has a pKa of about 3.5 to about7.5. In some embodiments, the buffer is phosphate, succinate, histidineor citrate. In certain embodiments, the buffer is succinate at a finalconcentration of 1 mM to 10 mM. In one particular embodiment, the finalconcentration of the succinate buffer is about 5 mM.

In an embodiment, the immunogenic compositions disclosed herein comprisea salt. In some embodiments, the salt is selected from the groupsconsisting of magnesium chloride, potassium chloride, sodium chlorideand a combination thereof. In one particular embodiment, the salt issodium chloride. In one particular embodiment, the immunogeniccompositions disclosed herein comprise sodium chloride at 150 mM.

In an embodiment, the immunogenic compositions disclosed herein comprisea surfactant. In an embodiment, the surfactant is selected from thegroup consisting of polysorbate 20 (TWEEN™20), polysorbate 40(TWEEN™40), polysorbate 60 (TWEEN™60), polysorbate 65 (TWEEN™65),polysorbate 80 (TWEEN™80), polysorbate 85 (TWEEN™85), TRITON™ N-101,TRITON™ X-100, oxtoxynol 40, nonoxynol-9, triethanolamine,triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate(PEG-15, Solutol H 15), polyoxyethylene-35-ricinoleate (CREMOPHOR® EL),soy lecithin and a poloxamer. In one particular embodiment, thesurfactant is polysorbate 80. In some said embodiment, the finalconcentration of polysorbate 80 in the formulation is at least 0.0001%to 10% polysorbate 80 weight to weight (w/w). In some said embodiments,the final concentration of polysorbate 80 in the formulation is at least0.001% to 1% polysorbate 80 weight to weight (w/w). In some saidembodiments, the final concentration of polysorbate 80 in theformulation is at least 0.01% to 1% polysorbate 80 weight to weight(w/w). In other embodiments, the final concentration of polysorbate 80in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%,0.08%, 0.09% or 0.1% polysorbate 80 (w/w). In another embodiment, thefinal concentration of the polysorbate 80 in the formulation is 1%polysorbate 80 (w/w).

In certain embodiments, the immunogenic composition disclosed herein hasa pH of 5.5 to 7.5, more preferably a pH of 5.6 to 7.0, even morepreferably a pH of 5.8 to 6.0.

In one embodiment, the present invention provides a container filledwith any of the immunogenic compositions disclosed herein. In oneembodiment, the container is selected from the group consisting of avial, a syringe, a flask, a fermentor, a bioreactor, a bag, a jar, anampoule, a cartridge and a disposable pen. In certain embodiments, thecontainer is siliconized.

In an embodiment, the container of the present invention is made ofglass, metals (e.g., steel, stainless steel, aluminum, etc.) and/orpolymers (e.g., thermoplastics, elastomers, thermoplastic-elastomers).In an embodiment, the container of the present invention is made ofglass.

In one embodiment, the present invention provides a syringe filled withany of the immunogenic compositions disclosed herein. In certainembodiments, the syringe is siliconized and/or is made of glass.

A typical dose of the immunogenic composition disclosed herein forinjection has a volume of 0.1 mL to 2 mL, more preferably 0.2 mL to 1mL, even more preferably a volume of about 0.5 mL.

Therefore the container or syringe as defined above is filed with avolume of 0.1 mL to 2 mL, more preferably 0.2 mL to 1 mL, even morepreferably a volume of about 0.5 mL of any of the immunogeniccompositions defined herein.

In an embodiment, the immunogenic composition of the invention (such asdefined at section 2 above) is formulated as disclosed above.

In an embodiment, the immunogenic composition which may be used incombination with the immunogenic composition of the invention (such asdefined at section 3 above) is formulated as disclosed above.

An aspect of the invention provides a kit as defined at section 4 abovewherein both immunogenic compositions (part (a) and (b) of the kit) areformulated as described above.

An aspect of the invention provides a kit as defined at section 4 abovewherein both immunogenic compositions (part (a) and (b) of the kit) areformulated in liquid form.

An aspect of the invention provides a kit as defined at section 4 abovewherein both immunogenic compositions (part (a) and (b) of the kit) areformulated in lyophilized form.

An aspect of the invention provides a kit as defined at section 4 abovewherein the first immunogenic composition (part (a) of the kit) is inliquid form and the second immunogenic composition (part (b) of the kit)is in lyophilized form.

An aspect of the invention provides a kit as defined at section 4 abovewherein the first immunogenic composition (part (a) of the kit) is inlyophilized form and the second immunogenic composition (part (b) of thekit) is in liquid form.

9. USES OF THE IMMUNOGENIC COMPOSITIONS AND KITS OF THE INVENTION

In an embodiment, the immunogenic compositions and kits disclosed hereinare for use as a medicament.

The immunogenic compositions and kits described herein may be used invarious therapeutic or prophylactic methods for preventing, treating orameliorating a bacterial infection, disease or condition in a subject.In particular, immunogenic compositions and kits described herein may beused to prevent, treat or ameliorate a S. pneumoniae infection, diseaseor condition in a subject.

Thus in one aspect, the invention provides a method of preventing,treating or ameliorating an infection, disease or condition associatedwith S. pneumoniae in a subject, comprising administering to the subjectan immunologically effective amount of an immunogenic composition ofdescribed herein.

In some such embodiments, the infection, disease or condition isselected from the group consisting of pneumonia, sinusitis, otitismedia, acute otitis media, meningitis, bacteremia, sepsis, pleuralempyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis,peritonitis, pericarditis, mastoiditis, cellulitis, soft tissueinfection and brain abscess.

In an embodiment, the invention provides a method of inducing an immuneresponse to S. pneumoniae in a subject comprising administering to thesubject an immunologically effective amount of an immunogeniccomposition of the invention

In an embodiment, the immunogenic compositions and kits disclosed hereinare for use as a vaccine. In such embodiments the immunogeniccompositions and kits described herein may be used to prevent a S.pneumoniae infection in a subject. Thus in one aspect, the inventionprovides a method of preventing an infection by S. pneumoniae in asubject comprising administering to the subject an immunologicallyeffective amount of an immunogenic composition of the invention. In somesuch embodiments, the infection is selected from the group consisting ofpneumonia, sinusitis, otitis media, acute otitis media, meningitis,bacteremia, sepsis, pleural empyema, conjunctivitis, osteomyelitis,septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis,cellulitis, soft tissue infection and brain abscess. In one aspect, thesubject to be vaccinated is a mammal, such as a human, cat, sheep, pig,horse, bovine or dog.

In one aspect, the immunogenic compositions and kits disclosed hereinare for use in a method of preventing, treating or ameliorating aninfection, disease or condition associated with S. pneumoniae in asubject. In some such embodiments, the infection, disease or conditionis selected from the group consisting of pneumonia, sinusitis, otitismedia, acute otitis media, meningitis, bacteremia, sepsis, pleuralempyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis,peritonitis, pericarditis, mastoiditis, cellulitis, soft tissueinfection and brain abscess.

In an embodiment, the immunogenic compositions and kits disclosed hereinare for use as a vaccine. In such embodiments the immunogeniccompositions and kits described herein may be used to prevent a S.pneumoniae infection in a subject. Thus in one aspect, the immunogeniccompositions and kits disclosed herein are for use in a method ofpreventing, an infection by S. pneumoniae in a subject. In some suchembodiments, the infection is selected from the group consisting ofpneumonia, sinusitis, otitis media, acute otitis media, meningitis,bacteremia, sepsis, pleural empyema, conjunctivitis, osteomyelitis,septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis,cellulitis, soft tissue infection and brain abscess. In one aspect, thesubject to be vaccinated is a mammal, such as a human, cat, sheep, pig,horse, bovine or dog.

The immunogenic compositions and kits of the present invention can beused to protect or treat a human susceptible to pneumococcal infection,by means of administering the immunogenic compositions via a systemic ormucosal route. In an embodiment, the immunogenic compositions disclosedherein are administered by intramuscular, intraperitoneal, intradermalor subcutaneous routes. In an embodiment, the immunogenic compositionsdisclosed herein are administered by intramuscular, intraperitoneal,intradermal or subcutaneous injection. In an embodiment, the immunogeniccompositions disclosed herein are administered by intramuscular orsubcutaneous injection.

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above), when administeredto a subject, are able to induce the formation of antibodies capable ofbinding to S. pneumonia serotype 15B, 15A and/or 15C as measured by astandard ELISA assay. In an embodiment, the immunogenic composition ofthe present disclosure comprising at least one glycoconjugate from S.pneumoniae serotype 15B (such as the glycoconjugates of section 1.3.4above), when administered to a subject, are able to induce the formationof antibodies capable of binding to S. pneumonia serotype 15B and 15C asmeasured by a standard ELISA assay.

In the ELISA (Enzyme-linked Immunosorbent Assay) method, antibodies fromthe sera of vaccinated subjects are incubated with polysaccharides whichhave been adsorbed to a solid support. The bound antibodies are detectedusing enzyme-conjugated secondary detection antibodies.

In an embodiment said standard ELISA assay is the standardized (WHO)ELISA assay as defined by the WHO in the ‘Training manual for Enzymelinked immunosorbent assay for the quantitation of Streptococcuspneumoniae serotype specific IgG (Pn PS ELISA).’ (accessible athttp://www.vaccine.uab.edu/ELISA %20protocol.pdf; last accessed on Mar.31, 2014).

The ELISA measures type specific IgG anti-S. pneumoniae capsularpolysaccharide (PS) antibodies present in human serum. When dilutions ofhuman sera are added to type-specific capsular PS-coated microtiterplates, antibodies specific for that capsular PS bind to the microtiterplates. The antibodies bound to the plates are detected using a goatanti-human IgG alkaline phosphatase-labeled antibody followed by ap-nitrophenyl phosphate substrate. The optical density of the coloredend product is proportional to the amount of anticapsular PS antibodypresent in the serum.

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above) is able to elicitIgG antibodies in human which are capable of binding S. pneumoniaeserotype 15B polysaccharide at a concentration of at least 0.05, 0.1,0.2, 0.3, 0.35, 0.4 or 0.5 μg/ml as determined by ELISA assay.

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above) is able to elicitIgG antibodies in human which are capable of binding S. pneumoniaeserotype 15C polysaccharide at a concentration of at least 0.05, 0.1,0.2, 0.3, 0.35, 0.4 or 0.5 μg/ml as determined by ELISA assay.

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above) is able to elicitIgG antibodies in human which are capable of binding S. pneumoniaeserotypes 15B and 15C polysaccharide at a concentration of at least0.05, 0.1, 0.2, 0.3, 0.35, 0.4 or 0.5 μg/ml as determined by ELISAassay.

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above), when administeredto a subject, are able to induce the formation of antibodies capable ofkilling S. pneumonia serotype 15B in an opsonophagocytosis assay asdisclosed herein (such as the OPA assay of Example 12).

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above), when tested in anOPA assay as disclosed herein (such as the OPA assay of Example 12), hasan OPA titer greater than the OPA titer obtained with an unconjugatednative S. pneumonia serotype 15B capsular polysaccharide.

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above), when administeredto a subject, are able to induce the formation of antibodies capable ofkilling S. pneumonia serotype 15C in an opsonophagocytosis assay asdisclosed herein (such as the OPA assay of Example 12). In anembodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above), when tested in anOPA assay as disclosed herein (such as the OPA assay of Example 12), hasan OPA titer greater than the OPA titer obtained with an unconjugatednative S. pneumonia serotype 15B capsular polysaccharide.

The pneumococcal opsonophagocytic assay (OPA), which measures killing ofS. pneumoniae cells by phagocytic effector cells in the presence offunctional antibody and complement, is considered to be an importantsurrogate for evaluating the effectiveness of pneumococcal vaccines.

Opsonophagocytic assay (OPA) can be conducted by incubating together amixture of Streptococcus pneumoniae cells, a heat inactivated humanserum to be tested, differentiated HL-60 cells (phagocytes) and anexogenous complement source (e.g. baby rabbit complement).Opsonophagocytosis proceeds during incubation and bacterial cells thatare coated with antibody and complement are killed uponopsonophagocytosis. Colony forming units (cfu) of surviving bacteriathat escape from opsonophagocytosis are determined by plating the assaymixture. The OPA titer is defined as the reciprocal dilution thatresults in a 50% reduction in bacterial count over control wells withouttest serum. The OPA titer is interpolated from the two dilutions thatencompass this 50% killing cut-off.

An endpoint titer of 1:8 or greater is considered a positive result inthese killing type OPA.

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above), is able to elicita titer of at least 1:8 against S. pneumoniae serotype 15B in at least50% of the subjects as determined by opsonophagocytic killing assay(OPA). In an embodiment, the immunogenic composition of the presentdisclosure comprising at least one glycoconjugate from S. pneumoniaeserotype 15B (such as the glycoconjugates of section 1.3.4 above) isable to elicit a titer of at least 1:8 against S. pneumoniae serotype15B in at least 60%, 70%, 80%, 90%, or at least 93% of the subjects asdetermined by opsonophagocytic killing assay (OPA).

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above) is able to elicit atiter of at least 1:8 against S. pneumoniae serotype 15C in at least 50%of the subjects as determined by opsonophagocytic killing assay (OPA).In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above) is able to elicit atiter of at least 1:8 against S. pneumoniae serotype 15C in at least60%, 70%, 80%, 90%, or at least 95% of the subjects as determined byopsonophagocytic killing assay (OPA).

In a further aspect, the present disclosure provides a method oftreating or preventing a S. pneumoniae infection, disease or conditionassociated with S. pneumoniae serotype 15A, 15B and/or 15C in a subject,the method comprising the step of administering a therapeutically orprophylactically effective amount of any of the immunogenic compositionsof the present disclosure comprising at least one glycoconjugate from S.pneumoniae serotype 15B (such as the glycoconjugates of section 1.3.4above). In an embodiment, the immunogenic composition of the presentdisclosure comprising at least one glycoconjugate from S. pneumoniaeserotype 15B (such as the glycoconjugates of section 1.3.4 above), whenadministered to a subject, induces the formation of antibodies capableof binding to S. pneumoniae serotype 15B, 15A and/or 15C. In anembodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above), when administeredto a subject, induces the formation of antibodies capable of killing S.pneumoniae serotype 15B, 15C and/or 15A in an opsonophagocytosis assayas disclosed herein (such as the OPA assay of Example 12).

One embodiment of the disclosure provides a method of protecting asubject against an infection with S. pneumoniae serotype 15C, or amethod of preventing infection with S. pneumoniae serotype 15C, or amethod of reducing the severity of or delaying the onset of at least onesymptom associated with an infection caused by S. pneumoniae serotype15C, the methods comprising administering to a subject an immunogenicamount of any of the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above). One embodiment ofthe disclosure provides a method of treating or preventing a S.pneumoniae infection, disease or condition associated with S. pneumoniaeserotype 15A, 15B and/or 15C (preferably 15B and/or 15C, more preferably15B) in a subject, the method comprising the step of administering atherapeutically or prophylactically effective amount of any of theimmunogenic composition of the present disclosure comprising at leastone glycoconjugate from S. pneumoniae serotype 15B (such as theglycoconjugates of section 1.3.4 above) to the subject. Anotherembodiment provides a method of treating or preventing a S. pneumoniaeinfection, disease or condition associated with a S. pneumoniae serotype15A, 15B and/or 15C (preferably 15B and/or 15C, more preferably 15B) ina subject, the method comprising generating a polyclonal or monoclonalantibody preparation from any of the immunogenic composition of thepresent disclosure comprising at least one glycoconjugate from S.pneumoniae serotype 15B (such as the glycoconjugates of section 1.3.4above), and using said antibody preparation to confer passive immunityto the subject.

In one embodiment, the disclosure relates to the use of any of theimmunogenic composition of the present disclosure comprising at leastone glycoconjugate from S. pneumoniae serotype 15B (such as theglycoconjugates of section 1.3.4 above) for the manufacture of amedicament for protecting a subject against an infection with S.pneumoniae, and/or preventing infection with S. pneumoniae, and/orreducing the severity of or delaying the onset of at least one symptomassociated with an infection caused by S. pneumoniae, and/or protectinga subject against an infection with S. pneumoniae serotype 15A, 15Band/or 15C (preferably 15B and/or 15C, more preferably 15B) and/orpreventing infection with S. pneumoniae serotype 15A, 15B and/or 15C(preferably 15B and/or 15C, more preferably 15B), and/or reducing theseverity of or delaying the onset of at least one symptom associatedwith an infection caused by S. pneumoniae serotype 15A, 15B and/or 15C(preferably 15B and/or 15C, more preferably 15B).

In one embodiment, the disclosure relates to the use of any of theimmunogenic composition of the present disclosure comprising at leastone glycoconjugate from S. pneumoniae serotype 15B (such as theglycoconjugates of section 1.3.4 above) for protecting a subject againstan infection with S. pneumoniae, and/or preventing infection with S.pneumoniae, and/or reducing the severity of or delaying the onset of atleast one symptom associated with an infection caused by S. pneumoniae,and/or protecting a subject against an infection with S. pneumoniaeserotype 15A, 15B and/or 15C (preferably 15B and/or 15C, more preferably15B) and/or preventing infection with S. pneumoniae serotype 15A, 15Band/or 15C (preferably 15B and/or 15C, more preferably 15B), and/orreducing the severity of or delaying the onset of at least one symptomassociated with an infection caused by S. pneumoniae serotype 15A, 15Band/or 15C (preferably 15B and/or 15C, more preferably 15B).

10. SUBJECT TO BE TREATED WITH THE IMMUNOGENIC COMPOSITIONS AND KITS OFTHE INVENTION

As disclosed herein, the immunogenic compositions and kits describedherein may be used in various therapeutic or prophylactic methods forpreventing, treating or ameliorating a bacterial infection, disease orcondition in a subject.

In a preferred embodiment, said subject is a human. In a most preferredembodiment, said subject is a newborn (i.e., under three months of age),an infant (i.e., from 3 months to one year of age) or a toddler (i.e.,from one year to four years of age).

In an embodiment, the immunogenic compositions and kits disclosed hereinare for use as a vaccine.

In such embodiment, the subject to be vaccinated may be less than 1 yearof age. For example, the subject to be vaccinated can be about 1, about2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about10, about 11 or about 12 months of age. In an embodiment, the subject tobe vaccinated is about 2, about 4 or about 6 months of age. In anotherembodiment, the subject to be vaccinated is less than 2 years of age.

For example the subject to be vaccinated can be about 12 to about 15months of age. In some cases, as little as one dose of the immunogeniccomposition according to the invention is needed, but under somecircumstances, a second, third or fourth dose may be given (see section11 below).

In an embodiment of the present invention, the subject to be vaccinatedis a human adult 50 years of age or older, more preferably a human adult55 years of age or older. In an embodiment, the subject to be vaccinatedis a human adult 65 years of age or older, 70 years of age or older, 75years of age or older or 80 years of age or older.

In an embodiment the subject to be vaccinated is an immunocompromisedindividual, in particular a human. An immunocompromised individual isgenerally defined as a person who exhibits an attenuated or reducedability to mount a normal humoral or cellular defense to challenge byinfectious agents.

In an embodiment of the present invention, the immunocompromised subjectto be vaccinated suffers from a disease or condition that impairs theimmune system and results in an antibody response that is insufficientto protect against or treat pneumococcal disease.

In an embodiment, said disease is a primary immunodeficiency disorder.Preferably, said primary immunodeficiency disorder is selected from thegroup consisting of: combined T- and B-cell immunodeficiencies, antibodydeficiencies, well-defined syndromes, immune dysregulation diseases,phagocyte disorders, innate immunity deficiencies, autoinflammatorydisorders, and complement deficiencies. In an embodiment, said primaryimmunodeficiency disorder is selected from the one disclosed on page 24,line 11, to page 25, line 19, of WO 2010/125480.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated suffers from a diseaseselected from the group consisting of: HIV-infection, acquiredimmunodeficiency syndrome (AIDS), cancer, chronic heart or lungdisorders, congestive heart failure, diabetes mellitus, chronic liverdisease, alcoholism, cirrhosis, spinal fluid leaks, cardiomyopathy,chronic bronchitis, emphysema, chronic obstructive pulmonary disease(COPD), spleen dysfunction (such as sickle cell disease), lack of spleenfunction (asplenia), blood malignancy, leukemia, multiple myeloma,Hodgkin's disease, lymphoma, kidney failure, nephrotic syndrome andasthma.

In an embodiment of the present invention, the immunocompromised subjectto be vaccinated suffers from malnutrition.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated is taking a drug or treatmentthat lowers the body's resistance to infection. In an embodiment, saiddrug is selected from the one disclosed on page 26, line 33, to page 26,line 4, of WO 2010/125480.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated is a smoker.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated has a white blood cell count(leukocyte count) below 5×10⁹ cells per liter, or below 4×10⁹ cells perliter, or below 3×10⁹ cells per liter, or below 2×10⁹ cells per liter,or below 1×10⁹ cells per liter, or below 0.5×10⁹ cells per liter, orbelow 0.3×10⁹ cells per liter, or below 0.1×10⁹ cells per liter.

White blood cell count (leukocyte count): The number of white bloodcells (WBC) in the blood. The WBC is usually measured as part of the CBC(complete blood count). White blood cells are the infection-fightingcells in the blood and are distinct from the red (oxygen-carrying) bloodcells known as erythrocytes. There are different types of white bloodcells, including neutrophils (polymorphonuclear leukocytes; PMN), bandcells (slightly immature neutrophils), T-type lymphocytes (T-cells),B-type lymphocytes (B-cells), monocytes, eosinophils, and basophils. Allthe types of white blood cells are reflected in the white blood cellcount. The normal range for the white blood cell count is usuallybetween 4,300 and 10,800 cells per cubic millimeter of blood. This canalso be referred to as the leukocyte count and can be expressed ininternational units as 4.3-10.8×10⁹ cells per liter.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated suffers from neutropenia. Ina particular embodiment of the present invention, the immunocompromisedsubject to be vaccinated has a neutrophil count below 2×10⁹ cells perliter, or below 1×10⁹ cells per liter, or below 0.5×10⁹ cells per liter,or below 0.1×10⁹ cells per liter, or below 0.05×10⁹ cells per liter.

A low white blood cell count or “neutropenia” is a conditioncharacterized by abnormally low levels of neutrophils in the circulatingblood. Neutrophils are a specific kind of white blood cell that help toprevent and fight infections. The most common reason that cancerpatients experience neutropenia is as a side effect of chemotherapy.Chemotherapy-induced neutropenia increases a patient's risk of infectionand disrupts cancer treatment.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated has a CD4+ cell count below500/mm³, or CD4+ cell count below 300/mm³, or CD4+ cell count below200/mm³, CD4+ cell count below 100/mm³, CD4+ cell count below 75/mm³, orCD4+ cell count below 50/mm³.

CD4 cell tests are normally reported as the number of cells in mm³.Normal CD4 counts are between 500 and 1,600, and CD8 counts are between375 and 1,100. CD4 counts drop dramatically in people with HIV.

In an embodiment of the invention, any of the immunocompromised subjectsdisclosed herein is a human male or a human female.

11. IMMUNIZATION SCHEDULE

In some cases, as little as one dose of the immunogenic compositionaccording to the invention is needed, but under some circumstances, suchas conditions of greater immune deficiency or immune immaturity, asecond, third or fourth dose may be given. Following an initialvaccination, subjects can receive one or several booster immunizationsadequately spaced.

In an embodiment, the schedule of vaccination of the immunogeniccomposition according to the invention is a single dose. In a particularembodiment, said single dose schedule is for healthy persons being atleast 2 years of age.

In an embodiment, the schedule of vaccination of the immunogeniccomposition according to the invention is a multiple dose schedule. Amultiple dose schedule is frequently used in conditions such as immunedeficiency (such as human elderly or human immunocompromisedindividuals) or immune immaturity (such as human newborns (i.e., underthree months of age), infants (i.e., from 3 months to one year of age)or toddlers (i.e., from one year to four years of age)). In a particularembodiment, said multiple dose schedule consists of a series of 2 dosesseparated by an interval of about 1 month to about 12 months. In aparticular embodiment, said multiple dose schedule consists of a seriesof 2 doses separated by an interval of about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 or 12 months. In a particular embodiment, said multiple doseschedule consists of a series of 2 doses separated by an interval ofabout 1 month to about 6 months. In a particular embodiment, saidmultiple dose schedule consists of a series of 2 doses separated by aninterval of about 1, 2, 3, 4, 5 or 6 months. In a particular embodiment,said multiple dose schedule consists of a series of 2 doses separated byan interval of about 1 month, or a series of 2 doses separated by aninterval of about 2 months.

In another embodiment, said multiple dose schedule consists of a seriesof 3 doses wherein each dose is separated by an interval of about 1month to about 12 months.

In a particular embodiment, said multiple dose schedule consists of aseries of 3 doses wherein each dose is separated by an interval of about1 month to about 6 months. In a particular embodiment, said multipledose schedule consists of a series of 3 doses wherein each dose isseparated by an interval of about 1, 2, 3, 4, 5 or 6 months. In aparticular embodiment, said multiple dose schedule consists of a seriesof 3 doses wherein each dose is separated by an interval of about 1month, or a series of 3 doses wherein each dose is separated by aninterval of about 2 months.

In another embodiment, said multiple dose schedule consists of a seriesof 4 doses wherein each dose is separated by an interval of about 1month to about 12 months.

In a particular embodiment, said multiple dose schedule consists of aseries of 4 doses wherein each dose is separated by an interval of about1 month to about 6 months. In a particular embodiment, said multipledose schedule consists of a series of 4 doses wherein each dose isseparated by an interval of about 1, 2, 3, 4, 5 or 6 months. In aparticular embodiment, said multiple dose schedule consists of a seriesof 4 doses wherein each dose is separated by an interval of about 1month, or a series of 4 doses wherein each dose is separated by aninterval of about 2 months.

In another embodiment, said multiple dose schedule consists of a seriesof 3 doses wherein each dose is separated by an interval of about 1month to about 4 months followed by a fourth dose about 10 months toabout 13 months after the first dose. In another embodiment, saidmultiple dose schedule consists of a series of 3 doses wherein each doseis separated by an interval of about 1, 2, 3 or 4 months followed by afourth dose about 10 months to about 13 months after the first dose. Inanother embodiment, said multiple dose schedule consists of a series of3 doses wherein each dose is separated by an interval of about 1 monthto about 2 months followed by a fourth dose about 10 months to about 13months after the first dose. In another embodiment, said multiple doseschedule consists of a series of 3 doses wherein each dose is separatedby an interval of about 1 month followed by a fourth dose about 10months to about 13 months after the first dose, or a series of 3 doseswherein each dose is separated by an interval of about 2 months followedby a fourth dose about 10 months to about 13 months after the firstdose.

In an embodiment, the multiple dose schedule consists of at least onedose (e.g., 1, 2 or 3 doses) in the first year of age followed by atleast one toddler dose.

In an embodiment, the multiple dose schedule consists of a series of 2or 3 doses wherein each dose is separated by an interval of about 1month to about 2 months (for example 28-56 days between doses), startingat 2 months of age, and followed by a toddler dose at 12-18 months ofage. In an embodiment, said multiple dose schedule consists of a seriesof 3 doses wherein each dose is separated by an interval of about 1month to about 2 months (for example 28-56 days between doses), startingat 2 months of age, and followed by a toddler dose at 12-15 months ofage. In another embodiment, said multiple dose schedule consists of aseries of 2 doses separated by an interval of about 2 months, startingat 2 months of age, and followed by a toddler dose at 12-18 months ofage.

In an embodiment, the multiple dose schedule consists of a 4-dose seriesof vaccine at 2, 4, 6, and 12-15 months of age.

In an embodiment, a prime dose is given at day 0 and one or more boosterdoses are given at intervals that range from about 2 to about 24 weeksbetween doses, preferably with a dosing interval of 4-8 weeks.

In an embodiment, a prime dose is given at day 0 and a boost is givenabout 3 months later.

In another embodiment, said multiple dose schedule consists of a seriesof 5 doses wherein each dose is separated by an interval of about 1month to about 12 months.

In a particular embodiment, said multiple dose schedule consists of aseries of 5 doses wherein each dose is separated by an interval of about1 month to about 6 months. In a particular embodiment, said multipledose schedule consists of a series of 5 doses wherein each dose isseparated by an interval of about 1, 2, 3, 4, 5 or 6 months. In aparticular embodiment, said multiple dose schedule consists of a seriesof 5 doses wherein each dose is separated by an interval of about 1month, or a series of 5 doses wherein each dose is separated by aninterval of about 2 months.

In another embodiment, said multiple dose schedule consists of a seriesof 6, 7 or 8 doses wherein each dose is separated by an interval ofabout 1 month to about 12 months. In a particular embodiment, saidmultiple dose schedule consists of a series of 6, 7 or 8 doses whereineach dose is separated by an interval of about 1 month to about 6months. In a particular embodiment, said multiple dose schedule consistsof a series of 6, 7 or 8 doses wherein each dose is separated by aninterval of about 1, 2, 3, 4, 5 or 6 months. In a particular embodiment,said multiple dose schedule consists of a series of 6, 7 or 8 doseswherein each dose is separated by an interval of about 1 month. In aparticular embodiment, said multiple dose schedule consists of a seriesof 6, 7 or 8 doses wherein each dose is separated by an interval ofabout 2 months.

An aspect of the invention pertains to any immunogenic composition ofthe invention for simultaneous, concurrent, concomitant or sequentialadministration with a second immunogenic composition. An aspect of theinvention pertains to any kit disclosed herein for simultaneous,concurrent, concomitant or sequential administration.

By “simultaneous administration” is meant the administration oftherapeutically effective doses of a first and a second immunogeniccompositions in a single unit dosage form.

By “concurrent administration” is meant the administration oftherapeutically effective doses of a first and a second immunogeniccompositions through the same access site, but in separate unit dosageforms, within a short period of one another. Concurrent administrationis essentially administering the two immunogenic compositions at aboutthe same time but in separate dosage forms, through the same accesssite. The concurrent administration of the first and the secondimmunogenic compositions often occurs during the same physician officevisit.

By “concomitant administration” is meant the administration oftherapeutically effective doses of a first and a second immunogeniccompositions, in separate unit dosage forms within a short period of oneanother at different anatomic sites. Concomitant administration isessentially administering the two immunogenic compositions at about thesame time but in separate dosage forms and at different anatomic sites.The concomitant administration of the first and second immunogeniccompositions often occurs during the same physician office visit.

By “sequential administration” is meant the administration of atherapeutically effective dose of a first or a second immunogeniccomposition alone, followed by the administration of a therapeuticallyeffective dose of the remaining immunogenic composition after aninterval of at least about 1 month. For instance in one embodiment, thefirst immunogenic composition is administered in a single dosage form,and then after an interval of at least about 1 month, the secondimmunogenic composition is administered in a separate single dosageform. In an alternative embodiment, the second immunogenic compositionis administered in a single dosage form, and then after an interval ofat least about 1 month, the first immunogenic composition isadministered in a separate single dosage form. The sequentialadministration of the first and second immunogenic compositions oftenoccurs at different physician office visits.

In an aspect of the present invention, a first immunogenic compositionaccording to the invention (such as the ones of section 2 above) isadministered simultaneously, concurrently, concomitantly or sequentiallywith a second immunogenic composition. In an embodiment said secondimmunogenic composition is any of the immunogenic compositions disclosedat section 3 above.

Therefore, an aspect of the present invention pertains to a firstimmunogenic composition according to the invention (such as the ones ofsection 2 above) for simultaneous, concurrent, concomitant or sequentialuse with a second immunogenic composition. In an embodiment said secondimmunogenic composition is any of the immunogenic compositions disclosedat section 3 above.

In some cases, as little as one dose of each of the immunogeniccompositions is needed, but under some circumstances, a second, third orfourth dose of one or each of the immunogenic composition may be given.Following an initial vaccination, subjects can receive one or severalbooster immunizations adequately spaced.

In an embodiment, the present invention pertains to a first immunogeniccomposition according to the invention (such as the ones of section 2above) for simultaneous administration with a second immunogeniccomposition. In an embodiment said second immunogenic composition is anyof the immunogenic compositions disclosed at section 3 above.

In an embodiment, the schedule of vaccination of said simultaneousadministration is a single dose. In a particular embodiment, said singledose schedule is for healthy persons being at least 2 years of age.

In an embodiment, the schedule of vaccination of said simultaneousadministration is a multiple dose schedule. In a particular embodiment,said multiple dose schedule consists of a series of 2 doses separated byan interval of about 1 month to about 12 months. In a particularembodiment, said multiple dose schedule consists of a series of 2 dosesseparated by an interval of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or12 months. In a particular embodiment, said multiple dose scheduleconsists of a series of 2 doses separated by an interval of about 1month to about 6 months. In a particular embodiment, said multiple doseschedule consists of a series of 2 doses separated by an interval ofabout 1, 2, 3, 4, 5 or 6 months. In a particular embodiment, saidmultiple dose schedule consists of a series of 2 doses separated by aninterval of about 1 month, or a series of 2 doses separated by aninterval of about 2 months.

In another embodiment, said multiple dose schedule consists of a seriesof 3 doses wherein each dose is separated by an interval of about 1month to about 12 months. In a particular embodiment, said multiple doseschedule consists of a series of 3 doses wherein each dose is separatedby an interval of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months.In a particular embodiment, said multiple dose schedule consists of aseries of 3 doses wherein each dose is separated by an interval of about1 month to about 6 months. In a particular embodiment, said multipledose schedule consists of a series of 3 doses wherein each dose isseparated by an interval of about 1, 2, 3, 4, 5 or 6 months. In anotherembodiment, said multiple dose schedule consists of a series of 3 doseswherein each dose is separated by an interval of about 1 month, or aseries of 3 doses wherein each dose is separated by an interval of about2 months.

In a particular embodiment, said multiple dose schedule consists of aseries of 4 doses separated by an interval of about 1 month to about 12months. In a particular embodiment, said multiple dose schedule consistsof a series of 4 doses separated by an interval of about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11 or 12 months. In a particular embodiment, saidmultiple dose schedule consists of a series of 4 doses separated by aninterval of about 1 month to about 6 months. In a particular embodiment,said multiple dose schedule consists of a series of 4 doses separated byan interval of about 1, 2, 3, 4, 5 or 6 months. In a particularembodiment, said multiple dose schedule consists of a series of 4 dosesseparated by an interval of about 1 month, or a series of 4 dosesseparated by an interval of about 2 months.

In another embodiment, said multiple dose schedule consists of a seriesof 3 doses wherein each dose is separated by an interval of about 1month to about 4 months followed by a fourth dose about 10 months toabout 13 months after the first dose. In another embodiment, saidmultiple dose schedule consists of a series of 3 doses wherein each doseis separated by an interval of about 1, 2, 3 or 4 months followed by afourth dose about 10 months to about 13 months after the first dose. Inanother embodiment, said multiple dose schedule consists of a series of3 doses wherein each dose is separated by an interval of about 1 monthto about 2 months followed by a fourth dose about 10 months to about 13months after the first dose. In another embodiment, said multiple doseschedule consists of a series of 3 doses wherein each dose is separatedby an interval of about 1 month followed by a fourth dose about 10months to about 13 months after the first dose, or a series of 3 doseswherein each dose is separated by an interval of about 2 months followedby a fourth dose about 10 months to about 13 months after the firstdose.

In an embodiment, the multiple dose schedule consists of at least onedose (e.g., 1, 2 or 3 doses) in the first year of age followed by atleast one toddler dose.

In an embodiment, the multiple dose schedule consists of a series of 2or 3 doses wherein each dose is separated by an interval of about 1month to about 2 months (for example 28-56 days between doses), startingat 2 months of age, and followed by a toddler dose at 12-18 months ofage. In an embodiment, said multiple dose schedule consists of a seriesof 3 doses wherein each dose is separated by an interval of about 1month to about 2 months (for example 28-56 days between doses), startingat 2 months of age, and followed by a toddler dose at 12-15 months ofage. In another embodiment, said multiple dose schedule consists of aseries of 2 doses separated by an interval of about 2 months, startingat 2 months of age, and followed by a toddler dose at 12-18 months ofage.

In an embodiment, the multiple dose schedule consists of a 4-dose seriesof vaccine administered at 2, 4, 6, and 12-15 months of age.

In an embodiment, a prime dose is given at day 0 and one or more boosterdoses are given at intervals that range from about 2 to about 24 weeksbetween doses, preferably with a dosing interval of 4-8 weeks.

In an embodiment, a prime dose is given at day 0 and a booster dose isgiven about 3 months later.

In a particular embodiment, said multiple dose schedule consists of aseries of 5, 6, 7 or 8 doses separated by an interval of about 1 monthto about 12 months. In a particular embodiment, said multiple doseschedule consists of a series of 5, 6, 7 or 8 doses separated by aninterval of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In aparticular embodiment, said multiple dose schedule consists of a seriesof 5, 6, 7 or 8 doses separated by an interval of about 1 month to about6 months. In a particular embodiment, said multiple dose scheduleconsists of a series of 5, 6, 7 or 8 doses separated by an interval ofabout 1, 2, 3, 4, 5 or 6 months. In a particular embodiment, saidmultiple dose schedule consists of a series of 5, 6, 7 or 8 dosesseparated by an interval of about 1 month, or a series of 5, 6, 7 or 8doses separated by an interval of about 2 months.

In an embodiment, the present invention pertains to a first immunogeniccomposition according to the invention (such as the ones of section 2above) for concomitant administration with a second immunogeniccomposition. In an embodiment said second immunogenic composition is anyof the immunogenic compositions disclosed at section 3 above.

In an embodiment, the schedule of vaccination of said concomitantadministration is a single dose (the administration of the first andsecond immunogenic composition, though in separate unit dosage forms, isconsidered as a single dose for purposes of defining the immunizationschedule). In a particular embodiment, said single dose schedule is forhealthy persons being at least 2 years of age.

In an embodiment, the schedule of vaccination of said concomitantadministration is a multiple dose schedule (the administration of thefirst and second immunogenic composition, though in separate unit dosageforms, is considered as a single dose for purposes of defining theimmunization schedule). In a particular embodiment, said multiple doseschedule consists of a series of 2 doses separated by an interval ofabout 1 month to about 12 months. In a particular embodiment, saidschedule consists of a series of 2 doses separated by an interval ofabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In a particularembodiment, said schedule consists of a series of 2 doses separated byan interval of about 1 month to about 6 months. In a particularembodiment, said schedule consists of a series of 2 doses separated byan interval of about 1, 2, 3, 4, 5 or 6 months. In a particularembodiment, said schedule consists of a series of 2 doses separated byan interval of about 1 month, or a series of 2 doses separated by aninterval of about 2 months.

In another embodiment, said multiple dose schedule consists of a seriesof 3 doses wherein each dose is separated by an interval of about 1month to about 12 months. In a particular embodiment, said scheduleconsists of a series of 3 doses wherein each dose is separated by aninterval of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In aparticular embodiment, said schedule consists of a series of 3 doseswherein each dose is separated by an interval of about 1 month to about6 months. In a particular embodiment, said schedule consists of a seriesof 3 doses wherein each dose is separated by an interval of about 1, 2,3, 4, 5 or 6 months. In another embodiment, said schedule consists of aseries of 3 doses wherein each dose is separated by an interval of about1 month, or a series of 3 doses wherein each dose is separated by aninterval of about 2 months.

In a particular embodiment, said multiple dose schedule consists of aseries of 4 doses separated by an interval of about 1 month to about 12months. In a particular embodiment, said multiple dose schedule consistsof a series of 4 doses separated by an interval of about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11 or 12 months. In a particular embodiment, saidmultiple dose schedule consists of a series of 4 doses separated by aninterval of about 1 month to about 6 months. In a particular embodiment,said multiple dose schedule consists of a series of 4 doses separated byan interval of about 1, 2, 3, 4, 5 or 6 months. In a particularembodiment, said multiple dose schedule consists of a series of 4 dosesseparated by an interval of about 1 month, or a series of 4 dosesseparated by an interval of about 2 months.

In another embodiment, said multiple dose schedule consists of a seriesof 3 doses wherein each dose is separated by an interval of about 1month to about 4 months followed by a fourth dose about 10 months toabout 13 months after the first dose. In another embodiment, saidschedule consists of a series of 3 doses wherein each dose is separatedby an interval of about 1, 2, 3 or 4 months followed by a fourth doseabout 10 months to about 13 months after the first dose. In anotherembodiment, said schedule consists of a series of 3 doses wherein eachdose is separated by an interval of about 1 month to about 2 monthsfollowed by a fourth dose about 10 months to about 13 months after thefirst dose. In another embodiment, said schedule consists of a series of3 doses wherein each dose is separated by an interval of about 1 monthfollowed by a fourth dose about 10 months to about 13 months after thefirst dose, or a series of 3 doses wherein each dose is separated by aninterval of about 2 months followed by a fourth dose about 10 months toabout 13 months after the first dose.

In an embodiment, the multiple dose schedule consists of at least onedose (e.g., 1, 2 or 3 doses) in the first year of age followed by atleast one toddler dose.

In an embodiment, the multiple dose schedule consists of a series of 2or 3 doses wherein each dose is separated by an interval of about 1month to about 2 months (for example 28-56 days between doses), startingat 2 months of age, and followed by a toddler dose at 12-18 months ofage. In an embodiment, said schedule consists of a series of 3 doseswherein each dose is separated by an interval of about 1 month to about2 months (for example 28-56 days between doses), starting at 2 months ofage, and followed by a toddler dose at 12-15 months of age. In anotherembodiment, said schedule consists of a series of 2 doses separated byan interval of about 2 months, starting at 2 months of age, and followedby a toddler dose at 12-18 months of age.

In an embodiment, the multiple dose schedule consists of a 4-dose seriesof vaccine administered at 2, 4, 6, and 12-15 months of age.

In an embodiment, a prime dose is given at day 0 and one or more boosterdoses are given at intervals that range from about 2 to about 24 weeks,preferably with a dosing interval of 4-8 weeks.

In an embodiment, a prime dose is given at day 0 and a boost is givenabout 3 months later.

In a particular embodiment, said multiple dose schedule consists of aseries of 5, 6, 7 or 8 doses separated by an interval of about 1 monthto about 12 months. In a particular embodiment, said multiple doseschedule consists of a series of 5, 6, 7 or 8 doses separated by aninterval of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In aparticular embodiment, said multiple dose schedule consists of a seriesof 5, 6, 7 or 8 doses separated by an interval of about 1 month to about6 months. In a particular embodiment, said multiple dose scheduleconsists of a series of 5, 6, 7 or 8 doses separated by an interval ofabout 1, 2, 3, 4, 5 or 6 months. In a particular embodiment, saidmultiple dose schedule consists of a series of 5, 6, 7 or 8 dosesseparated by an interval of about 1 month, or a series of 5, 6, 7 or 8doses separated by an interval of about 2 months.

In another embodiment, the present invention pertains to a firstimmunogenic composition according to the invention (such as the ones ofsection 2 above) for concurrent administration with a second immunogeniccomposition. In an embodiment said second immunogenic composition is anyof the immunogenic compositions disclosed at section 3 above.

In an embodiment, the schedule of vaccination of said concurrentadministration is a single dose (the administration of the first andsecond immunogenic composition, though in separate unit dosage forms, isconsidered as a single dose for purposes of defining the immunizationschedule). In a particular embodiment, said single dose schedule is forhealthy persons being at least 2 years of age.

In an embodiment, the schedule of vaccination of said concurrentadministration is a multiple dose schedule, in particular any of themultiple schedules disclosed above for a concomitant administration.

In an embodiment, the present invention pertains to a first immunogeniccomposition according to the invention (such as the ones of section 2above) for sequential administration with a second immunogeniccomposition. In an embodiment said second immunogenic composition is anyof the immunogenic compositions disclosed at section 3 above.

In an embodiment, the first immunogenic composition according to theinvention is administered first and the second immunogenic compositionis administered second. In another embodiment, the second immunogeniccomposition is administered first and the first immunogenic compositionaccording to the invention is administered second.

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 2, 3, 4, 5, 6, 7 or 8 doses.

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 2, 3 or 4 doses

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 2 doses. In an embodiment, theschedule of vaccination of said sequential administration consists of aseries of 2 doses separated by an interval of about 1 month to about 12months. In a particular embodiment, said multiple dose schedule consistsof a series of 2 doses separated by an interval of about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11 or 12 months. In a particular embodiment, saidmultiple dose schedule consists of a series of 2 doses separated by aninterval of about 1 month to about 6 months. In a particular embodiment,said multiple dose schedule consists of a series of 2 doses separated byan interval of about 1, 2, 3, 4, 5 or 6 months. In a particularembodiment, said multiple dose schedule consists of a series of 2 dosesseparated by an interval of about 1 month, or a series of 2 dosesseparated by an interval of about 2 months.

In an embodiment of said 2-dose schedule, the first immunogeniccomposition according to the invention is administered first and thesecond immunogenic composition is administered second. In anotherembodiment, the second immunogenic composition is administered first andthe first immunogenic composition according to the invention isadministered second.

In an embodiment of said 2-dose schedule, the first and second doses areadministered in the first year of age. In an embodiment of said 2-doseschedules, the first dose is administered in the first year of age andthe second dose is a toddler dose. In an embodiment, said toddler doseis administered at 12-18 months of age. In an embodiment, said toddlerdose is administered at 12-15 months of age.

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 3 doses. In a particularembodiment, said schedule consists of a series of 3 doses wherein eachdose is separated by an interval of about 1 to about 12 months. In aparticular embodiment, said schedule consists of a series of 3 doseswherein each dose is separated by an interval of about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11 or 12 months. In a particular embodiment, said scheduleconsists of a series of 3 doses wherein each dose is separated by aninterval of about 1 month to about 6 months. In a particular embodiment,said schedule consists of a series of 3 doses wherein each dose isseparated by an interval of about 1, 2, 3, 4, 5 or 6 months. In aparticular embodiment, said schedule consists of a series of 3 doseswherein each dose is separated by an interval of about 1 to about 2months. In another embodiment, said schedule consists of a series of 3doses wherein each dose is separated by an interval of about 1 month, ora series of 3 doses wherein each dose is separated by an interval ofabout 2 months.

In an embodiment of said 3-dose schedule, the first and second doses areadministered in the first year of age and the third dose is a toddlerdose. In an embodiment, the first and second doses are separated by aninterval of about 1 month to about 2 months (for example 28-56 daysbetween doses), starting at 2 months of age, and the third dose is atoddler dose at 12-18 months of age. In an embodiment, the first andsecond doses are separated by an interval of about 1 month to about 2months (for example 28-56 days between doses), starting at 2 months ofage, and the third dose is a toddler dose at 12-15 months of age.

In an embodiment of said 3-dose schedule, the first immunogeniccomposition according to the invention is administered as the first twodoses and the second immunogenic composition is administered as thethird dose.

In another embodiment of said 3-dose schedule, the second immunogeniccomposition is administered as the first two doses and the firstimmunogenic composition according to the invention is administered asthe third dose.

In another embodiment of said 3-dose schedule, the first immunogeniccomposition according to the invention is administered as the firstdose, the second immunogenic composition is administered as the seconddose and the first immunogenic composition according to the invention isadministered as the third dose.

In yet another embodiment of said 3-dose schedule, the secondimmunogenic composition is administered as the first dose, the firstimmunogenic composition according to the invention is administered asthe second dose and the second immunogenic composition is administeredas the third dose.

In yet another embodiment of said 3-dose schedule, the first immunogeniccomposition according to the invention is administered as the first doseand the second immunogenic composition is administered as the second andthird doses.

In another embodiment of said 3-dose schedule, the second immunogeniccomposition is administered as the first dose and the first immunogeniccomposition according to the invention is administered as the second andthird doses.

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 4 doses.

In a particular embodiment, said schedule consists of a series of 4doses wherein each dose is separated by an interval of about 1 to about12 months. In a particular embodiment, said schedule consists of aseries of 4 doses wherein each dose is separated by an interval of about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In a particularembodiment, said schedule consists of a series of 4 doses wherein eachdose is separated by an interval of about 1 month to about 6 months. Ina particular embodiment, said schedule consists of a series of 4 doseswherein each dose is separated by an interval of about 1, 2, 3, 4, 5 or6 months. In a particular embodiment, said schedule consists of a seriesof 4 doses wherein each dose is separated by an interval of about 1 toabout 2 months. In another embodiment, said schedule consists of aseries of 4 doses wherein each dose is separated by an interval of about1 month, or a series of 4 doses wherein each dose is separated by aninterval of about 2 months.

In an embodiment of said 4-dose schedule, said schedule consists of aseries of 3 doses wherein each dose is separated by an interval of about1 month to about 4 months followed by a fourth dose about 10 months toabout 13 months after the first dose. In another embodiment, saidschedule consists of a series of 3 doses wherein each dose is separatedby an interval of about 1, 2, 3 or 4 months followed by a fourth doseabout 10 months to about 13 months after the first dose. In anotherembodiment, said schedule consists of a series of 3 doses wherein eachdose is separated by an interval of about 1 month to about 2 monthsfollowed by a fourth dose about 10 months to about 13 months after thefirst dose. In another embodiment, said schedule consists of a series of3 doses wherein each dose is separated by an interval of about 1 monthfollowed by a fourth dose about 10 months to about 13 months after thefirst dose, or a series of 3 doses wherein each dose is separated by aninterval of about 2 months followed by a fourth dose about 10 months toabout 13 months after the first dose.

In another embodiment, said schedule consists of a series of 2 doseswherein each dose is separated by an interval of about 1 month to about2 months followed by a third dose about 10 months to about 13 monthsafter the first dose and a fourth dose about 1 month to about 2 monthsafter the third dose.

In an embodiment of said 4-dose schedule the first and second doses areadministered in the first year of age and the third and fourth doses area toddler dose.

In an embodiment of said 4-dose schedule the first, second and thirddoses are administered in the first year of age and the fourth dose is atoddler dose.

In an embodiment, said 4-dose schedule consists of a series of 3 doseswherein each dose is separated by an interval of about 1 month to about2 months (for example 28-56 days between doses), starting at 2 months ofage, followed by a toddler dose at 12-18 months of age. In anembodiment, said schedule consists of a series of 3 doses wherein eachdose is separated by an interval of about 1 month to about 2 months (forexample 28-56 days between doses), starting at 2 months of age, followedby a toddler dose at 12-15 months of age.

In an embodiment, the multiple dose schedule consists of a 4-dose seriesof vaccine at 2, 4, 6, and 12-15 months of age.

In an embodiment of said 4-dose schedule, the first immunogeniccomposition according to the invention is administered as the firstthree doses and the second immunogenic composition is administered asthe fourth dose.

In another embodiment of said 4-dose schedule, the second immunogeniccomposition is administered as the first three doses and the firstimmunogenic composition according to the invention is administered asthe fourth dose.

In another embodiment of said 4-dose schedule, the first immunogeniccomposition according to the invention is administered as the first andsecond doses and the second immunogenic composition is administered asthe third and fourth doses.

In another embodiment of said 4-dose schedule, the second immunogeniccomposition is administered as the first and second doses and the firstimmunogenic composition according to the invention is administered asthe third and fourth doses.

In another embodiment of said 4-dose schedule, the first immunogeniccomposition according to the invention is administered as the first andsecond doses, the second immunogenic composition is administered as thethird dose and the first immunogenic composition according to theinvention is administered as the fourth dose.

In another embodiment of said 4-dose schedule, the second immunogeniccomposition is administered as the first and second doses, the firstimmunogenic composition according to the invention is administered asthe third dose and the second immunogenic composition is administered asthe fourth dose.

In another embodiment of said 4-dose schedule, the first immunogeniccomposition according to the invention is administered as the first doseand the second immunogenic composition is administered as the second,third and fourth doses.

In another embodiment of said 4-dose schedule, the second immunogeniccomposition is administered as the first dose and the first immunogeniccomposition according to the invention is administered as the second,third and fourth doses.

In another embodiment of said 4-dose schedule, the first immunogeniccomposition according to the invention is administered as the firstdose, the second immunogenic composition is administered as the seconddose, the first immunogenic composition according to the invention isadministered as the third dose and the second immunogenic composition isadministered as the fourth dose.

In another embodiment of said 4-dose schedule, the second immunogeniccomposition is administered as the first dose, the first immunogeniccomposition according to the invention is administered as the seconddose, the second immunogenic composition is administered as the thirddose and the first immunogenic composition according to the invention isadministered as the fourth dose.

In another embodiment of said 4-dose schedule, the first immunogeniccomposition according to the invention is administered as the firstdose, the second immunogenic composition is administered as the seconddose and the first immunogenic composition according to the invention isadministered as the third and fourth doses.

In another embodiment of said 4-dose schedule, the second immunogeniccomposition is administered as the first dose, the first immunogeniccomposition according to the invention is administered as the seconddose and the second immunogenic composition is administered as the thirdand fourth doses.

In another embodiment of said 4-dose schedule, the first immunogeniccomposition according to the invention is administered as the firstdose, the second immunogenic composition is administered as the secondand third doses and the first immunogenic composition according to theinvention is administered as the fourth dose.

In another embodiment of said 4-dose schedule, the second immunogeniccomposition is administered as the first dose, the first immunogeniccomposition according to the invention is administered as the second andthird doses and the second immunogenic composition is administered asthe fourth dose.

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 5 doses.

In a particular embodiment, said schedule consists of a series of 5doses wherein each dose is separated by an interval of about 1 to about12 months. In a particular embodiment, said schedule consists of aseries of 5 doses wherein each dose is separated by an interval of about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In a particularembodiment, said schedule consists of a series of 5 doses wherein eachdose is separated by an interval of about 1 month to about 6 months. Ina particular embodiment, said schedule consists of a series of 5 doseswherein each dose is separated by an interval of about 1, 2, 3, 4, 5 or6 months. In a particular embodiment, said schedule consists of a seriesof 5 doses wherein each dose is separated by an interval of about 1 toabout 2 months. In another embodiment, said schedule consists of aseries of 5 doses wherein each dose is separated by an interval of about1 month, or a series of 5 doses wherein each dose is separated by aninterval of about 2 months.

In an embodiment said 5-dose schedule consists of a series of 4 doseswherein each dose is separated by an interval of about 1 month to about3 months followed by a fifth dose about 10 months to about 13 monthsafter the first dose. In another embodiment, said schedule consists of aseries of 4 doses wherein each dose is separated by an interval of about1 month to about 2 months followed by a fifth dose about 10 months toabout 13 months after the first dose. In another embodiment, saidschedule consists of a series of 4 doses wherein each dose is separatedby an interval of about 1 month followed by a fifth dose about 10 monthsto about 13 months after the first dose, or a series of 4 doses whereineach dose is separated by an interval of about 2 months followed by afifth dose about 10 months to about 13 months after the first dose.

In another embodiment, said schedule consists of a series of 3 doseswherein each dose is separated by an interval of about 1 month to about2 months followed by a fourth dose about 10 months to about 13 monthsafter the first dose and a fifth dose about 1 month to about 2 monthsafter the fourth dose.

In an embodiment of said 5-doses schedule the first, second and thirddoses are administered in the first year of age and the fourth and fifthdoses are a toddler dose.

In an embodiment of said 5-doses schedule, the first, second, third andfourth doses are administered in the first year of age and the fifthdose is a toddler dose. In an embodiment, said 5-doses schedule consistsof a series of 4 doses wherein each dose is separated by an interval ofabout 1 month to about 2 months (for example 28-56 days between doses),starting at 2 months of age, and followed by a toddler dose at 12-18months of age. In an embodiment, said schedule consists of a series of 4doses wherein each dose is separated by an interval of about 1 month toabout 2 months (for example 28-56 days between doses), starting at 2months of age, and followed by a toddler dose at 12-15 months of age.

In an embodiment of said 5-doses schedule, the first immunogeniccomposition according to the invention (such as the ones of section 2above, designated 1^(st) IC in the below table) and the secondimmunogenic composition (such as disclosed at section 3 above,designated 2^(nd) IC in the below table) are administered in thefollowing order:

Dose Schedule number 1 2 3 4 5 1 2^(nd) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC1^(st) IC 2 2^(nd) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 3 2^(nd)IC 2^(nd) IC 2^(nd) IC 1^(st) IC 1^(st) IC 4 2^(nd) IC 2^(nd) IC 1^(st)IC 2^(nd) IC 2^(nd) IC 5 2^(nd) IC 2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd)IC 6 2^(nd) IC 2^(nd) IC 1^(st) IC 1^(st) IC 1^(st) IC 7 2^(nd) IC2^(nd) IC 1^(st) IC 2^(nd) IC 1^(st) IC 8 2^(nd) IC 1^(st) IC 2^(nd) IC2^(nd) IC 2^(nd) IC 9 2^(nd) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC10 2^(nd) IC 1^(st) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 11 2^(nd) IC 1^(st)IC 2^(nd) IC 1^(st) IC 1^(st) IC 12 2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd)IC 2^(nd) IC 13 2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd) IC 1^(st) IC 142^(nd) IC 1^(st) IC 1^(st) IC 1^(st) IC 2^(nd) IC 15 2^(nd) IC 1^(st) IC1^(st) IC 1^(st) IC 1^(st) IC 16 1^(st) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC2^(nd) IC 17 1^(st) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 18 1^(st)IC 2^(nd) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 19 1^(st) IC 2^(nd) IC 2^(nd)IC 1^(st) IC 1^(st) IC 20 1^(st) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 2^(nd)IC 21 1^(st) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 1^(st) IC 22 1^(st) IC2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd) IC 23 1^(st) IC 2^(nd) IC 1^(st) IC1^(st) IC 1^(st) IC 24 1^(st) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC25 1^(st) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 26 1^(st) IC 1^(st)IC 2^(nd) IC 1^(st) IC 2^(nd) IC 27 1^(st) IC 1^(st) IC 2^(nd) IC 1^(st)IC 1^(st) IC 28 1^(st) IC 1^(st) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 291^(st) IC 1^(st) IC 1^(st) IC 2^(nd) IC 1^(st) IC 30 1^(st) IC 1^(st) IC1^(st) IC 1^(st) IC 2^(nd) IC

The above table provide the order of administration of the first andsecond immunogenic composition (designated 1^(st) IC and 2^(nd) ICrespectively) for the different doses, for example schedule number 1 isto be read as: in embodiment of said 5-dose schedule, the secondimmunogenic composition is administered as the first, second, third andfourth doses and the first immunogenic composition according to theinvention is administered as the fifth dose.

In a preferred embodiment, the order of administration of the first andsecond immunogenic composition is according to schedule 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 18, 19, 20 or 21.

In an embodiment, the schedule of vaccination of said sequential doseconsists of a series of 6 doses.

In a particular embodiment, said schedule consists of a series of 6doses wherein each dose is separated by an interval of about 1 to about12 months. In a particular embodiment, said schedule consists of aseries of 6 doses wherein each dose is separated by an interval of about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In a particularembodiment, said schedule consists of a series of 6 doses wherein eachdose is separated by an interval of about 1 month to about 6 months. Ina particular embodiment, said schedule consists of a series of 6 doseswherein each dose is separated by an interval of about 1, 2, 3, 4, 5 or6 months. In a particular embodiment, said schedule consists of a seriesof 6 doses wherein each dose is separated by an interval of about 1 toabout 2 months. In another embodiment, said schedule consists of aseries of 6 doses wherein each dose is separated by an interval of about1 month, or a series of 6 doses wherein each dose is separated by aninterval of about 2 months.

In an embodiment said 6-dose schedule consists of a series of 5 doseswherein each dose is separated by an interval of about 1 month to about2 months followed by a sixth dose about 10 months to about 13 monthsafter the first dose. In another embodiment, said schedule consists of aseries of 5 doses wherein each dose is separated by an interval of about1 month followed by a sixth dose about 10 months to about 13 monthsafter the first dose, or a series of 5 doses wherein each dose isseparated by an interval of about 2 months followed by a sixth doseabout 10 months to about 13 months after the first dose.

In an embodiment of said 6-doses schedule, the first, second, third,fourth and fifth doses are administered in the first year of age and thesixth dose is a toddler dose. In an embodiment, said 6-doses scheduleconsists of a series of 5 doses wherein each dose is separated by aninterval of about 1 month to about 2 months (for example 28-56 daysbetween doses), starting at 2 months of age, and followed by a toddlerdose at 12-18 months of age. In an embodiment, said schedule consists ofa series of 5 doses wherein each dose is separated by an interval ofabout 1 month to about 2 months (for example 28-56 days between doses),starting at 2 months of age, and followed by a toddler dose at 12-15months of age.

In an embodiment of said 6-doses schedule, the first immunogeniccomposition according to the invention (such as the ones of section 2above) and the second immunogenic composition (such as disclosed atsection 3 above) are administered in the order according to the any ofthe 30 schedules provided for the 5-doses schedule (see above table,schedule 1 to 30), followed by a sixth dose. In an embodiment, the firstimmunogenic composition according to the invention is administered asthe sixth dose. In another embodiment, the second immunogeniccomposition is administered as the sixth dose.

In an embodiment, the schedule of vaccination of said sequential doseconsists of a series of 7 doses.

In a particular embodiment, said schedule consists of a series of 7doses wherein each dose is separated by an interval of about 1 to about12 months. In a particular embodiment, said schedule consists of aseries of 7 doses wherein each dose is separated by an interval of about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In a particularembodiment, said schedule consists of a series of 7 doses wherein eachdose is separated by an interval of about 1 month to about 6 months. Ina particular embodiment, said schedule consists of a series of 7 doseswherein each dose is separated by an interval of about 1, 2, 3, 4, 5 or6 months. In a particular embodiment, said schedule consists of a seriesof 7 doses wherein each dose is separated by an interval of about 1 toabout 2 months. In another embodiment, said schedule consists of aseries of 7 doses wherein each dose is separated by an interval of about1 month, or a series of 7 doses wherein each dose is separated by aninterval of about 2 months.

In an embodiment said 7-dose schedule consists of a series of 6 doseswherein each dose is separated by an interval of about 1 month followedby a seventh dose about 10 months to about 13 months after the firstdose.

In an embodiment of said 7-doses schedule, the first, second, third,fourth, fifth and sixth doses are administered in the first year of ageand the seventh dose is a toddler dose. In an embodiment, said 7-doseschedule consists of a series of 6 doses wherein each dose is separatedby an interval of about 1 month (for example 28-40 days between doses),starting at 2 months of age, and followed by a toddler dose at 12-18months of age. In an embodiment, said schedule consists of a series of 6doses wherein each dose is separated by an interval of about 1 month(for example 28-40 days between doses), starting at 2 months of age, andfollowed by a toddler dose at 12-15 months of age.

In an embodiment of said 7-doses schedule, the first immunogeniccomposition according to the invention (such as the ones of section 2above) and the second immunogenic composition (such as disclosed atsection 3 above) are administered in the order according to the any ofthe schedules provided for the 6-doses schedule (see above), followed bya seventh dose. In an embodiment, the first immunogenic compositionaccording to the invention is administered as the seventh dose. Inanother embodiment, the second immunogenic composition is administeredas the seventh dose.

In an embodiment, the schedule of vaccination of said sequential doseconsists of a series of 8 doses.

In a particular embodiment, said schedule consists of a series of 8doses wherein each dose is separated by an interval of about 1 to about12 months. In a particular embodiment, said schedule consists of aseries of 8 doses wherein each dose is separated by an interval of about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In a particularembodiment, said schedule consists of a series of 8 doses wherein eachdose is separated by an interval of about 1 month to about 6 months. Ina particular embodiment, said schedule consists of a series of 8 doseswherein each dose is separated by an interval of about 1, 2, 3, 4, 5 or6 months. In a particular embodiment, said schedule consists of a seriesof 8 doses wherein each dose is separated by an interval of about 1 toabout 2 months. In another embodiment, said schedule consists of aseries of 8 doses wherein each dose is separated by an interval of about1 month, or a series of 8 doses wherein each dose is separated by aninterval of about 2 months.

In an embodiment said 8-dose schedule consists of a series of 7 doseswherein each dose is separated by an interval of about 1 month followedby an eighth dose about 10 months to about 13 months after the firstdose.

In an embodiment of said 8-doses schedule, the first, second, third,fourth, fifth, sixth and seventh doses are administered in the firstyear of age and the eighth dose is a toddler dose. In an embodiment,said 8-dose schedule consists of a series of 7 doses wherein each doseis separated by an interval of about 1 month (for example 28-40 daysbetween doses), starting at 2 months of age, and followed by a toddlerdose at 12-18 months of age. In an embodiment, said schedule consists ofa series of 7 doses wherein each dose is separated by an interval ofabout 1 month (for example 28-40 days between doses), starting at 2months of age, and followed by a toddler dose at 12-15 months of age.

In an embodiment of said 8-doses schedule, the first immunogeniccomposition according to the invention (such as the ones of section 2above) and the second immunogenic composition (such as disclosed atsection 3 above) are administered in the order according to the any ofthe schedules provided for the 7-doses schedule (see above), followed bya eighth dose. In an embodiment, the first immunogenic compositionaccording to the invention is administered as the eighth dose. Inanother embodiment, the second immunogenic composition is administeredas the eighth dose.

In an embodiment, the present invention pertains to the sequentialadministration of:

-   -   (a) a first immunogenic composition according to the invention        (such as the ones of section 2 above) and    -   (b) the concomitant administration of the first immunogenic        composition according to the invention (such as the ones of        section 2 above) with a second immunogenic composition.

In an embodiment said second immunogenic composition is any of theimmunogenic compositions disclosed at section 3 above.

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 2 administrations. In anembodiment, the schedule of vaccination consists of a series of 2administrations separated by an interval of about 1 month to about 12months. In a particular embodiment, said schedule consists of a seriesof 2 administrations separated by an interval of about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11 or 12 months. In a particular embodiment, said scheduleconsists of a series of 2 administrations separated by an interval ofabout 1 month to about 6 months. In a particular embodiment, saidschedule consists of a series of 2 administrations separated by aninterval of about 1, 2, 3, 4, 5 or 6 months. In an embodiment, theschedule of vaccination consists of a series of 2 administrationsseparated by an interval of about 1 month to about 2 months. In aparticular embodiment, said schedule consists of a series of 2administrations separated by an interval of about 1 month, or a seriesof 2 administrations separated by an interval of about 2 months.

In an embodiment of said schedule, a first immunogenic compositionaccording to the invention is administered first and the concomitantadministration of the first immunogenic composition according to theinvention with a second immunogenic composition is administered second.In another embodiment, the concomitant administration of a firstimmunogenic composition according to the invention with asecondimmunogenic composition is administered first and the first immunogeniccomposition according to the invention is administered second.

In an embodiment of said 2-administrations schedule, the first andsecond administrations are administered in the first year of age. In anembodiment of said 2-administrations schedule, the first administrationis administered in the first year of age and the second administrationis a toddler administration. In an embodiment, said toddleradministration is administered at 12-18 months of age. In an embodiment,said toddler administration is administered at 12-15 months of age.

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 3 administrations. In anembodiment, said schedule consists of a series of 3 administrationsseparated by an interval of about 1 month to about 12 months. In aparticular embodiment, said schedule consists of a series of 3administrations wherein each administration is separated by an intervalof about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In a particularembodiment, said schedule consists of a series of 3 administrationswherein each administration is separated by an interval of about 1 monthto about 6 months. In a particular embodiment, said schedule consists ofa series of 3 administrations wherein each administration is separatedby an interval of about 1, 2, 3, 4, 5 or 6 months. In an embodiment,said schedule consists of a series of 3 administrations separated by aninterval of about 1 month to about 2 months. In another embodiment, saidschedule consists of a series of 3 administrations wherein eachadministration is separated by an interval of about 1 month, or a seriesof 3 administrations wherein each administration is separated by aninterval of about 2 months.

In an embodiment of said 3-administrations schedule, the first andsecond administrations are administered in the first year of age and thethird administration is a toddler administration. In an embodiment, thefirst and second administrations are separated by an interval of about 1month to about 2 months (for example 28-56 days betweenadministrations), starting at 2 months of age, and the thirdadministration is a toddler administration at 12-18 months of age. In anembodiment, the first and second administrations are separated by aninterval of about 1 month to about 2 months (for example 28-56 daysbetween administrations), starting at 2 months of age, and the thirdadministration is a toddler administration at 12-15 months of age.

In an embodiment of said 3-administrations schedule, the firstimmunogenic composition according to the invention is administered atthe first and second administrations and the concomitant administrationof the first immunogenic composition according to the invention with thesecond immunogenic composition is administered at the thirdadministration.

In another embodiment of said 3-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention with the second immunogenic composition isadministered at the first and second administrations and the firstimmunogenic composition according to the invention is administered atthe third administration.

In another embodiment of said 3-administrations schedule, the firstimmunogenic composition according to the invention is administered atthe first administration, the concomitant administration of the firstimmunogenic composition according to the invention with the secondimmunogenic composition is administered at the second administration andthe first immunogenic composition according to the invention isadministered at the third administration.

In yet another embodiment of said 3-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention with the second immunogenic composition isadministered at the first administration, the first immunogeniccomposition according to the invention is administered at the secondadministration and the concomitant administration of the firstimmunogenic composition according to the invention with the secondimmunogenic composition is administered at the third administration.

In yet another embodiment of said 3-administrations schedule, the firstimmunogenic composition according to the invention is administered atthe first administration and the concomitant administration of the firstimmunogenic composition according to the invention with the secondimmunogenic composition is administered at the second and thirdadministrations.

In another embodiment of said 3-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention with the second immunogenic composition isadministered at the first administration and the first immunogeniccomposition according to the invention is administered at the second andthird administrations.

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 4 administrations.

In an embodiment, said schedule consists of a series of 4administrations separated by an interval of about 1 month to about 12months. In a particular embodiment, said schedule consists of a seriesof 4 administrations wherein each administration is separated by aninterval of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In aparticular embodiment, said schedule consists of a series of 4administrations wherein each administration is separated by an intervalof about 1 month to about 6 months. In a particular embodiment, saidschedule consists of a series of 4 administrations wherein eachadministration is separated by an interval of about 1, 2, 3, 4, 5 or 6months. In an embodiment, said schedule consists of a series of 4administrations separated by an interval of about 1 month to about 2months. In another embodiment, said schedule consists of a series of 4administrations wherein each administration is separated by an intervalof about 1 month, or a series of 4 administrations wherein eachadministration is separated by an interval of about 2 months.

In an embodiment of said 4-administrations schedule, said scheduleconsists of a series of 3 administrations wherein each administration isseparated by an interval of about 1 month to about 4 months followed bya fourth administration about 10 months to about 13 months after thefirst administration. In another embodiment, said schedule consists of aseries of 3 administrations wherein each administration is separated byan interval of about 1, 2, 3 or 4 months followed by a fourthadministration about 10 months to about 13 months after the firstadministration. In another embodiment, said schedule consists of aseries of 3 administrations wherein each administration is separated byan interval of about 1 month to about 2 months followed by a fourthadministration about 10 months to about 13 months after the firstadministration. In another embodiment, said schedule consists of aseries of 3 administrations wherein each administration is separated byan interval of about 1 month followed by a fourth administration about10 months to about 13 months after the first administration, or a seriesof 3 administrations wherein each administration is separated by aninterval of about 2 months followed by a fourth administration about 10months to about 13 months after the first administration.

In an embodiment of said 4-administrations schedule, the first, secondand third administrations are administered in the first year of age andthe fourth administration is a toddler administration. In an embodiment,said 4-administrations schedule consists of a series of 3administrations wherein each administration is separated by an intervalof about 1 month to about 2 months (for example 28-56 days betweenadministrations), starting at 2 months of age, and followed by a toddleradministration at 12-18 months of age. In an embodiment, said scheduleconsists of a series of 3 administrations wherein each administration isseparated by an interval of about 1 month to about 2 months (for example28-56 days between administrations), starting at 2 months of age, andfollowed by a toddler administration at 12-15 months of age.

In an embodiment, said 4-administrations schedule consists of a seriesof administrations at 2, 4, 6, and 12-15 months of age.

In an embodiment of said 4-administrations schedule, the firstimmunogenic composition according to the invention is administered atthe first, second and third administrations and the concomitantadministration of the first immunogenic composition according to theinvention with the second immunogenic composition is administered at thefourth administration.

In another embodiment of said 4-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention with the second immunogenic composition isadministered at the first, second and third administrations and thefirst immunogenic composition according to the invention is administeredat the fourth administration.

In another embodiment of said 4-administrations schedule, the firstimmunogenic composition according to the invention is administered atthe first and second administrations and the concomitant administrationof the first immunogenic composition according to the invention with thesecond immunogenic composition is administered at the third and fourthadministrations.

In another embodiment of said 4-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention with the second immunogenic composition isadministered at the first and second administrations and the firstimmunogenic composition according to the invention is administered atthe third and fourth administrations.

In another embodiment of said 4-administrations schedule, the firstimmunogenic composition according to the invention is administered atthe first and second administrations, the concomitant administration ofthe first immunogenic composition according to the invention with thesecond immunogenic composition is administered at the thirdadministration and the first immunogenic composition according to theinvention is administered at the fourth administration.

In another embodiment of said 4-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention with the second immunogenic composition isadministered at the first and second administrations, the firstimmunogenic composition according to the invention is administered atthe third administration and the concomitant administration of the firstimmunogenic composition according to the invention with the secondimmunogenic composition is administered at the fourth administration.

In another embodiment of said 4-administrations schedule, the firstimmunogenic composition according to the invention is administered atthe first administration and the concomitant administration of the firstimmunogenic composition according to the invention with the secondimmunogenic composition is administered at the second, third and fourthadministrations.

In another embodiment of said 4-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention with the second immunogenic composition isadministered at the first administration and the first immunogeniccomposition according to the invention is administered at the second,third and fourth administration.

In another embodiment of said 4-administrations schedule, the firstimmunogenic composition according to the invention is administered atthe first administration, the concomitant administration of the firstimmunogenic composition according to the invention with the secondimmunogenic composition is administered at the second administration,the first immunogenic composition according to the invention isadministered at the third administration and the concomitantadministration of the first immunogenic composition according to theinvention with the second immunogenic composition is administered at thefourth administration.

In another embodiment of said 4-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention with the second immunogenic composition isadministered at the first administration, the first immunogeniccomposition according to the invention is administered at the secondadministration, the concomitant administration of the first immunogeniccomposition according to the invention with the second immunogeniccomposition is administered at the third administration and the firstimmunogenic composition according to the invention is administered atthe fourth administration.

In another embodiment of said 4-administrations schedule, the firstimmunogenic composition according to the invention is administered atthe first administration, the concomitant administration of the firstimmunogenic composition according to the invention with the secondimmunogenic composition is administered at the second administration andthe first immunogenic composition according to the invention isadministered at the third and fourth administrations.

In another embodiment of said 4-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention with the second immunogenic composition isadministered at the first administration, the first immunogeniccomposition according to the invention is administered at the secondadministration and the concomitant administration of the firstimmunogenic composition according to the invention with the secondimmunogenic composition is administered at the third and fourthadministrations.

In another embodiment of said 4-administrations schedule, the firstimmunogenic composition according to the invention is administered atthe first administration, the concomitant administration of the firstimmunogenic composition according to the invention with the secondimmunogenic composition is administered at the second and thirdadministrations and the first immunogenic composition according to theinvention is administered at the fourth administration.

In another embodiment of said 4-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention with the second immunogenic composition isadministered at the first administration, the first immunogeniccomposition according to the invention is administered at the second andthird administrations and the concomitant administration of the firstimmunogenic composition according to the invention with the secondimmunogenic composition is administered at the fourth administration.

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 5 administrations.

In an embodiment, said schedule consists of a series of 5administrations separated by an interval of about 1 month to about 12months. In a particular embodiment, said schedule consists of a seriesof 5 administrations wherein each administration is separated by aninterval of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In aparticular embodiment, said schedule consists of a series of 5administrations wherein each administration is separated by an intervalof about 1 month to about 6 months. In a particular embodiment, saidschedule consists of a series of 5 administrations wherein eachadministration is separated by an interval of about 1, 2, 3, 4, 5 or 6months. In an embodiment, said schedule consists of a series of 5administrations separated by an interval of about 1 month to about 2months. In another embodiment, said schedule consists of a series of 5administrations wherein each administration is separated by an intervalof about 1 month, or a series of 5 administrations wherein eachadministration is separated by an interval of about 2 months.

In an embodiment said schedule consists of a series of 4 administrationswherein each dose is separated by an interval of about 1 month to about3 months followed by a fifth administration about 10 months to about 13months after the first administration. In another embodiment, saidschedule consists of a series of 4 administrations wherein eachadministration is separated by an interval of about 1 month to about 2months followed by a fifth administration about 10 months to about 13months after the first dose. In another embodiment, said scheduleconsists of a series of 4 administrations wherein each dose is separatedby an interval of about 1 month followed by a fifth administration about10 months to about 13 months after the first administration, or a seriesof 4 administrations wherein each administration is separated by aninterval of about 2 months followed by a fifth administration about 10months to about 13 months after the first administration.

In an embodiment of said 5-administrations schedule, the first, second,third and fourth administrations are administered in the first year ofage and the fifth administration is a toddler dose. In an embodiment,said 5-administrations schedule consists of a series of 4administrations wherein each administration is separated by an intervalof about 1 month to about 2 months (for example 28-56 days betweendoses), starting at 2 months of age, and followed by a toddleradministration at 12-18 months of age. In an embodiment, said scheduleconsists of a series of 4 administrations wherein each administrationsis separated by an interval of about 1 month to about 2 months (forexample 28-56 days between doses), starting at 2 months of age, andfollowed by a toddler administration at 12-15 months of age.

In an embodiment of said 5-administrations schedule, the firstimmunogenic composition according to the invention (designated 1^(st) ICin the below table) and the concomitant administration of the firstimmunogenic composition according to the invention with the secondimmunogenic composition (designated 1^(st) IC/2^(nd) IC in the belowtable A) are administered in the following order:

TABLE A Schedule Dose number 1 2 3 4 5 1 1st IC/2nd IC 1st IC/2nd IC 1stIC/2nd IC 1st IC/2nd IC 1^(st) IC 2 1st IC/2nd IC 1st IC/2nd IC 1stIC/2nd IC 1^(st) IC 1st IC/2nd IC 3 1st IC/2nd IC 1st IC/2nd IC 1stIC/2nd IC 1^(st) IC 1^(st) IC 4 1st IC/2nd IC 1st IC/2nd IC 1^(st) IC1st IC/2nd IC 1st IC/2nd IC 5 1st IC/2nd IC 1st IC/2nd IC 1^(st) IC1^(st) IC 1st IC/2nd IC 6 1st IC/2nd IC 1st IC/2nd IC 1^(st) IC 1^(st)IC 1^(st) IC 7 1st IC/2nd IC 1st IC/2nd IC 1^(st) IC 1st IC/2nd IC1^(st) IC 8 1st IC/2nd IC 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 1stIC/2nd IC 9 1st IC/2nd IC 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 1^(st)IC 10 1st IC/2nd IC 1^(st) IC 1st IC/2nd IC 1^(st) IC 1st IC/2nd IC 111st IC/2nd IC 1^(st) IC 1st IC/2nd IC 1^(st) IC 1^(st) IC 12 1st IC/2ndIC 1^(st) IC 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 13 1st IC/2nd IC1^(st) IC 1^(st) IC 1st IC/2nd IC 1^(st) IC 14 1st IC/2nd IC 1^(st) IC1^(st) IC 1^(st) IC 1st IC/2nd IC 15 1st IC/2nd IC 1^(st) IC 1^(st) IC1^(st) IC 1^(st) IC 16 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 1st IC/2ndIC 1st IC/2nd IC 17 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC1^(st) IC 18 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 1^(st) IC 1st IC/2ndIC 19 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 1^(st) IC 1^(st) IC 201^(st) IC 1st IC/2nd IC 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 21 1^(st)IC 1st IC/2nd IC 1^(st) IC 1st IC/2nd IC 1^(st) IC 22 1^(st) IC 1stIC/2nd IC 1^(st) IC 1^(st) IC 1st IC/2nd IC 23 1^(st) IC 1st IC/2nd IC1^(st) IC 1^(st) IC 1^(st) IC 24 1^(st) IC 1^(st) IC 1st IC/2nd IC 1stIC/2nd IC 1st IC/2nd IC 25 1^(st) IC 1^(st) IC 1st IC/2nd IC 1st IC/2ndIC 1^(st) IC 26 1^(st) IC 1^(st) IC 1st IC/2nd IC 1^(st) IC 1st IC/2ndIC 27 1^(st) IC 1^(st) IC 1st IC/2nd IC 1^(st) IC 1^(st) IC 28 1^(st) IC1^(st) IC 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 29 1^(st) IC 1^(st) IC1^(st) IC 1st IC/2nd IC 1^(st) IC 30 1^(st) IC 1^(st) IC 1^(st) IC1^(st) IC 1st IC/2nd IC

The above table provide the order of administration of the firstimmunogenic composition according to the invention (designated 1^(st) ICin the below table) and the concomitant administration of the firstimmunogenic composition according to the invention with the secondimmunogenic composition (designated 1^(st) IC/2^(nd) IC in the belowtable) for the different doses, for example schedule number 1 is to beread as: in embodiment of said 5-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention with the second immunogenic composition isadministered as the first, second, third and fourth doses and the firstimmunogenic composition according to the invention is administered asthe fifth dose. In a preferred embodiment, the order of administrationis according to schedule 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,16, 17, 18, 19, 20, 22 or 23.

In an embodiment, the schedule of vaccination of said sequential doseconsists of a series of 6 administrations.

In an embodiment, said schedule consists of a series of 6administrations separated by an interval of about 1 month to about 12months. In a particular embodiment, said schedule consists of a seriesof 6 administrations wherein each administration is separated by aninterval of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In aparticular embodiment, said schedule consists of a series of 6administrations wherein each administration is separated by an intervalof about 1 month to about 6 months. In a particular embodiment, saidschedule consists of a series of 6 administrations wherein eachadministration is separated by an interval of about 1, 2, 3, 4, 5 or 6months. In an embodiment, said schedule consists of a series of 6administrations separated by an interval of about 1 month to about 2months. In another embodiment, said schedule consists of a series of 6administrations wherein each administration is separated by an intervalof about 1 month, or a series of 6 administrations wherein eachadministration is separated by an interval of about 2 months.

In an embodiment said 6-administrations schedule consists of a series of5 administrations wherein each administration is separated by aninterval of about 1 month to about 2 months followed by a sixthadministration about 10 months to about 13 months after the firstadministration. In another embodiment, said schedule consists of aseries of 5 administrations wherein each administration is separated byan interval of about 1 month followed by a sixth administration about 10months to about 13 months after the first administration, or a series of5 administrations wherein each administration is separated by aninterval of about 2 months followed by a sixth administration about 10months to about 13 months after the first administration.

In an embodiment of said 6-administrations schedule, the first, second,third, fourth and fifth administrations are administered in the firstyear of age and the sixth administration is a toddler administration. Inan embodiment, said 6-administrations schedule consists of a series of 5administrations wherein each administration is separated by an intervalof about 1 month to about 2 months (for example 28-56 days betweenadministrations), starting at 2 months of age, and followed by a toddleradministration at 12-18 months of age. In an embodiment, said scheduleconsists of a series of 5 administrations wherein each administration isseparated by an interval of about 1 month to about 2 months (for example28-56 days between administrations), starting at 2 months of age, andfollowed by a toddler administration at 12-15 months of age.

In an embodiment of said 6-administrations schedule, the firstimmunogenic composition according to the invention and the concomitantadministration of the first immunogenic composition according to theinvention with the second immunogenic composition are administered inthe order according to the any of the 30 schedules provided for the5-administrations schedule (see above table, schedule 1 to 30), followedby a sixth administration. In an embodiment, the first immunogeniccomposition according to the invention is administered at the sixthadministration. In another embodiment, the concomitant administration ofthe first immunogenic composition according to the invention with thesecond immunogenic composition is administered at the sixthadministration.

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 7 administrations.

In an embodiment, said schedule consists of a series of 7administrations separated by an interval of about 1 month to about 12months. In a particular embodiment, said schedule consists of a seriesof 7 administrations wherein each administration is separated by aninterval of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In aparticular embodiment, said schedule consists of a series of 7administrations wherein each administration is separated by an intervalof about 1 month to about 6 months. In a particular embodiment, saidschedule consists of a series of 7 administrations wherein eachadministration is separated by an interval of about 1, 2, 3, 4, 5 or 6months. In an embodiment, said schedule consists of a series of 7administrations separated by an interval of about 1 month to about 2months. In another embodiment, said schedule consists of a series of 7administrations wherein each administration is separated by an intervalof about 1 month, or a series of 7 administrations wherein eachadministration is separated by an interval of about 2 months.

In an embodiment said 7-administrations schedule consists of a series of6 administrations wherein each administration is separated by aninterval of about 1 month followed by a seventh administration about 10months to about 13 months after the first administration.

In an embodiment of said 7-administrations schedule, the first, second,third, fourth, fifth and sixth administrations are administered in thefirst year of age and the seventh administration is a toddleradministration. In an embodiment, said 7-administrations scheduleconsists of a series of 6 administrations wherein each administration isseparated by an interval of about 1 month (for example 28-40 daysbetween administrations), starting at 2 months of age, and followed by atoddler administration at 12-18 months of age. In an embodiment, saidschedule consists of a series of 6 administrations wherein eachadministration is separated by an interval of about 1 month (for example28-40 days between administrations), starting at 2 months of age, andfollowed by a toddler administration at 12-15 months of age.

In an embodiment of said 7-administrations schedule, the firstimmunogenic composition according to the invention (such as the ones ofsection 2 above) and the concomitant administration of the firstimmunogenic composition according to the invention with the secondimmunogenic composition are administered in the order according to theany of the schedules provided for the 6-administrations schedule (seeabove), followed by a seventh administration. In an embodiment, thefirst immunogenic composition according to the invention is administeredat the seventh administration. In another embodiment, the concomitantadministration of the first immunogenic composition according to theinvention with the second immunogenic composition is administered at theseventh administration.

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 8 administrations.

In an embodiment, said schedule consists of a series of 8administrations separated by an interval of about 1 month to about 12months. In a particular embodiment, said schedule consists of a seriesof 8 administrations wherein each administration is separated by aninterval of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In aparticular embodiment, said schedule consists of a series of 8administrations wherein each administration is separated by an intervalof about 1 month to about 6 months. In a particular embodiment, saidschedule consists of a series of 8 administrations wherein eachadministration is separated by an interval of about 1, 2, 3, 4, 5 or 6months. In an embodiment, said schedule consists of a series of 8administrations separated by an interval of about 1 month to about 2months. In another embodiment, said schedule consists of a series of 8administrations wherein each administration is separated by an intervalof about 1 month, or a series of 8 administrations wherein eachadministration is separated by an interval of about 2 months.

In an embodiment said 8-administration schedule consists of a series of7 administrations wherein each administration is separated by aninterval of about 1 month followed by an eight administration about 10months to about 13 months after the first administration.

In an embodiment of said 8-administrations schedule, the first, second,third, fourth, fifth, sixth and seventh administrations are administeredin the first year of age and the eighth administration is a toddleradministration. In an embodiment, said 8-administrations scheduleconsists of a series of 7 administrations wherein each administration isseparated by an interval of about 1 month (for example 28-40 daysbetween administrations), starting at 2 months of age, and followed by atoddler administration at 12-18 months of age. In an embodiment, saidschedule consists of a series of 7 administrations wherein eachadministration is separated by an interval of about 1 month (for example28-40 days between administrations), starting at 2 months of age, andfollowed by a toddler administration at 12-15 months of age.

In an embodiment of said 8-administrations schedule, the firstimmunogenic composition according to the invention (such as the ones ofsection 2 above) and the concomitant administration of the firstimmunogenic composition according to the invention with the secondimmunogenic composition are administered in the order according to theany of the schedules provided for the 7-administrations schedule (seeabove), followed by a eighth dose. In an embodiment, the firstimmunogenic composition according to the invention is administered atthe eighth dose. In another embodiment, the concomitant administrationof the first immunogenic composition according to the invention with thesecond immunogenic composition is administered at the eighth dose.

In an embodiment, in the administration schedules disclosed above theconcomitant administration(s) is/are replaced by a concurrentadministration.

In an embodiment, the present invention pertains to the sequentialadministration of:

-   -   (a) the second immunogenic composition (such as the ones of        section 3 above) and    -   (b) the concomitant administration of the first immunogenic        composition according to the invention (such as the ones of        section 2 above) with said second immunogenic composition.

In an embodiment said second immunogenic composition is any of theimmunogenic compositions disclosed at section 3 above.

In an embodiment, the schedule of administration is any one of theschedules disclosed above for the sequential administration of a firstimmunogenic composition according to the invention and the concomitantadministration of the first immunogenic composition according to theinvention with a second immunogenic composition (bottom of page 152- topage 164), wherein administration of said second immunogenic compositionof (a) replace administration of the first immunogenic composition of(a) in said schedules.

In an embodiment, in any of the administration schedules disclosed abovea concomitant administration(s) is/are replaced by a concurrentadministration.

Therefore in an embodiment, the present invention pertains to thesequential administration of:

-   -   (a) the second immunogenic composition (such as the ones of        section 3 above) and    -   (b) the concomitant administration of the first immunogenic        composition according to the invention (such as the ones of        section 2 above) with said second immunogenic composition.

In an embodiment said second immunogenic composition is any of theimmunogenic compositions disclosed at section 3 above.

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 2 administrations. In anembodiment, the schedule of vaccination consists of a series of 2administrations separated by an interval of about 1 month to about 12months. In a particular embodiment, said schedule consists of a seriesof 2 administrations separated by an interval of about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11 or 12 months. In a particular embodiment, said scheduleconsists of a series of 2 administrations separated by an interval ofabout 1 month to about 6 months. In a particular embodiment, saidschedule consists of a series of 2 administrations separated by aninterval of about 1, 2, 3, 4, 5 or 6 months. In an embodiment, theschedule of vaccination consists of a series of 2 administrationsseparated by an interval of about 1 month to about 2 months. In aparticular embodiment, said schedule consists of a series of 2administrations separated by an interval of about 1 month, or a seriesof 2 administrations separated by an interval of about 2 months.

In an embodiment of said schedule, the second immunogenic composition(such as the ones of section 3 above) is administered first and theconcomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) withsaid second immunogenic composition is administered second. In anotherembodiment, the concomitant administration of the first immunogeniccomposition according to the invention (such as the ones of section 2above) with said second immunogenic composition is administered firstand the second immunogenic composition (such as the ones of section 3above) is administered second.

In an embodiment of said 2-administrations schedule, the first andsecond administrations are administered in the first year of age. In anembodiment of said 2-administrations schedule, the first administrationis administered in the first year of age and the second administrationis a toddler administration. In an embodiment, said toddleradministration is administered at 12-18 months of age. In an embodiment,said toddler administration is administered at 12-15 months of age.

In an embodiment, in any of the 2-administrations schedules disclosedabove the concomitant administration(s) is/are replaced by a concurrentadministration.

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 3 administrations. In anembodiment, said schedule consists of a series of 3 administrationsseparated by an interval of about 1 month to about 12 months. In aparticular embodiment, said schedule consists of a series of 3administrations wherein each administration is separated by an intervalof about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In a particularembodiment, said schedule consists of a series of 3 administrationswherein each administration is separated by an interval of about 1 monthto about 6 months. In a particular embodiment, said schedule consists ofa series of 3 administrations wherein each administration is separatedby an interval of about 1, 2, 3, 4, 5 or 6 months. In an embodiment,said schedule consists of a series of 3 administrations separated by aninterval of about 1 month to about 2 months. In another embodiment, saidschedule consists of a series of 3 administrations wherein eachadministration is separated by an interval of about 1 month, or a seriesof 3 administrations wherein each administration is separated by aninterval of about 2 months. In another embodiment, said scheduleconsists of a series of 3 administrations wherein the first twoadministrations are separated by an interval of about 1 month to about 2months followed by a third administration about 10 months to about 13months after the first administration.

In an embodiment of said 3-administrations schedule, the first andsecond administrations are administered in the first year of age and thethird administration is a toddler administration. In an embodiment, thefirst and second administrations are separated by an interval of about 1month to about 2 months (for example 28-56 days betweenadministrations), starting at 2 months of age, and the thirdadministration is a toddler administration at 12-18 months of age. In anembodiment, the first and second administrations are separated by aninterval of about 1 month to about 2 months (for example 28-56 daysbetween administrations), starting at 2 months of age, and the thirdadministration is a toddler administration at 12-15 months of age.

In an embodiment of said schedule, the second immunogenic composition(such as the ones of section 3 above) is administered first and theconcomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) withsaid second immunogenic composition is administered second.

In an embodiment of said 3-administrations schedule, the secondimmunogenic composition (such as the ones of section 3 above) isadministered at the first and second administrations and the concomitantadministration of the first immunogenic composition according to theinvention (such as the ones of section 2 above) with said secondimmunogenic composition is administered at the third administration.

In another embodiment of said 3-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) withsaid second immunogenic composition is administered at the first andsecond administrations and the second immunogenic composition (such asthe ones of section 3 above) is administered at the thirdadministration.

In another embodiment of said 3-administrations schedule, the secondimmunogenic composition (such as the ones of section 3 above) isadministered at the first administration, the concomitant administrationof the first immunogenic composition according to the invention (such asthe ones of section 2 above) is administered at the secondadministration and the second immunogenic composition (such as the onesof section 3 above) is administered at the third administration.

In yet another embodiment of said 3-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) isadministered at the first administration, the second immunogeniccomposition (such as the ones of section 3 above) is administered at thesecond administration and the concomitant administration of the firstimmunogenic composition according to the invention (such as the ones ofsection 2 above) is administered at the third administration.

In yet another embodiment of said 3-administrations schedule, the secondimmunogenic composition (such as the ones of section 3 above) isadministered at the first administration and the concomitantadministration of the first immunogenic composition according to theinvention (such as the ones of section 2 above) is administered at thesecond and third administrations.

In another embodiment of said 3-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) isadministered at the first administration and the second immunogeniccomposition (such as the ones of section 3 above) is administered at thesecond and third administrations.

Therefore in an embodiment of said 3-administrations schedule, thesecond immunogenic composition (such as the ones of section 3 above)(designated 2^(nd) IC in the below table) and the concomitantadministration of the first immunogenic composition according to theinvention (such as the ones of section 2 above) (designated 1^(st)IC/2^(nd) IC in the below table B) are administered in the followingorder:

TABLE B Schedule Dose number 1 2 3 1 2^(nd) IC 2^(nd) IC 1st IC/2nd IC 21st IC/2nd IC 1st IC/2nd IC 2^(nd) IC 3 2^(nd) IC 1st IC/2nd IC 2^(nd)IC 4 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 5 2^(nd) IC 1st IC/2nd IC 1stIC/2nd IC 6 1st IC/2nd IC 2^(nd) IC 2^(nd) IC

The above table provides the order of administration of the secondimmunogenic composition (such as the ones of section 3 above)(designated 2^(nd) IC in the below table) and the concomitantadministration of the first immunogenic composition according to theinvention with said second immunogenic composition (designated 1^(st)IC/2^(nd) IC in the below table) for the different doses, for exampleschedule number 1 is to be read as: in embodiment of said3-administrations schedule, the second immunogenic composition isadministered as the first and second doses and the concomitantadministration of the first immunogenic composition according to theinvention with said second immunogenic composition is administered asthe third dose.

In a preferred embodiment, the order of administration is according toschedule 1.

In an embodiment, in any of the 3-administrations schedules disclosedabove the concomitant administration(s) is/are replaced by a concurrentadministration.

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 4 administrations.

In an embodiment, said schedule consists of a series of 4administrations separated by an interval of about 1 month to about 12months. In a particular embodiment, said schedule consists of a seriesof 4 administrations wherein each administration is separated by aninterval of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In aparticular embodiment, said schedule consists of a series of 4administrations wherein each administration is separated by an intervalof about 1 month to about 6 months. In a particular embodiment, saidschedule consists of a series of 4 administrations wherein eachadministration is separated by an interval of about 1, 2, 3, 4, 5 or 6months. In an embodiment, said schedule consists of a series of 4administrations separated by an interval of about 1 month to about 2months. In another embodiment, said schedule consists of a series of 4administrations wherein each administration is separated by an intervalof about 1 month, or a series of 4 administrations wherein eachadministration is separated by an interval of about 2 months.

In an embodiment of said 4-administrations schedule, said scheduleconsists of a series of 3 administrations wherein each administration isseparated by an interval of about 1 month to about 4 months followed bya fourth administration about 10 months to about 13 months after thefirst administration. In another embodiment, said schedule consists of aseries of 3 administrations wherein each administration is separated byan interval of about 1, 2, 3 or 4 months followed by a fourthadministration about 10 months to about 13 months after the firstadministration. In another embodiment, said schedule consists of aseries of 3 administrations wherein each administration is separated byan interval of about 1 month to about 2 months followed by a fourthadministration about 10 months to about 13 months after the firstadministration. In another embodiment, said schedule consists of aseries of 3 administrations wherein each administration is separated byan interval of about 1 month followed by a fourth administration about10 months to about 13 months after the first administration, or a seriesof 3 administrations wherein each administration is separated by aninterval of about 2 months followed by a fourth administration about 10months to about 13 months after the first administration.

In an embodiment of said 4-administrations schedule, the first, secondand third administrations are administered in the first year of age andthe fourth administration is a toddler administration. In an embodiment,said 4-administrations schedule consists of a series of 3administrations wherein each administration is separated by an intervalof about 1 month to about 2 months (for example 28-56 days betweenadministrations), starting at 2 months of age, and followed by a toddleradministration at 12-18 months of age. In an embodiment, said scheduleconsists of a series of 3 administrations wherein each administration isseparated by an interval of about 1 month to about 2 months (for example28-56 days between administrations), starting at 2 months of age, andfollowed by a toddler administration at 12-15 months of age.

In an embodiment, said 4-administrations schedule consists of a seriesof administrations at 2, 4, 6, and 12-15 months of age.

In an embodiment of said 4-administrations schedule, the secondimmunogenic composition (such as the ones of section 3 above) isadministered at the first, second and third administrations and theconcomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) withsaid second immunogenic composition is administered at the fourthadministration.

In another embodiment of said 4-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) withsaid second immunogenic composition is administered at the first, secondand third administrations and the second immunogenic composition (suchas the ones of section 3 above) is administered at the fourthadministration.

In another embodiment of said 4-administrations schedule, the secondimmunogenic composition (such as the ones of section 3 above) isadministered at the first and second administrations and the concomitantadministration of the first immunogenic composition according to theinvention (such as the ones of section 2 above) with said secondimmunogenic composition is administered at the third and fourthadministrations.

In another embodiment of said 4-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) withsaid second immunogenic composition is administered at the first andsecond administrations and the second immunogenic composition (such asthe ones of section 3 above) is administered at the third and fourthadministrations.

In another embodiment of said 4-administrations schedule, the secondimmunogenic composition (such as the ones of section 3 above) isadministered at the first and second administrations, the concomitantadministration of the first immunogenic composition according to theinvention (such as the ones of section 2 above) with said secondimmunogenic composition is administered at the third administration andthe second immunogenic composition (such as the ones of section 3 above)is administered at the fourth administration.

In another embodiment of said 4-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) withsaid second immunogenic composition is administered at the first andsecond administrations, the second immunogenic composition (such as theones of section 3 above) is administered at the third administration andthe concomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) withsaid second immunogenic composition is administered at the fourthadministration.

In another embodiment of said 4-administrations schedule, the secondimmunogenic composition (such as the ones of section 3 above) isadministered at the first administration and the concomitantadministration of the first immunogenic composition according to theinvention (such as the ones of section 2 above) with said secondimmunogenic composition is administered at the second, third and fourthadministrations.

In another embodiment of said 4-administration schedule, the concomitantadministration of the first immunogenic composition according to theinvention (such as the ones of section 2 above) with said secondimmunogenic composition is administered at the first administration andthe second immunogenic composition (such as the ones of section 3 above)is administered at the second, third and fourth administration.

In another embodiment of said 4-administrations schedule, the secondimmunogenic composition (such as the ones of section 3 above) isadministered at the first administration, the concomitant administrationof the first immunogenic composition according to the invention (such asthe ones of section 2 above) with said second immunogenic composition isadministered at the second administration, the second immunogeniccomposition (such as the ones of section 3 above) is administered at thethird administration and the concomitant administration of the firstimmunogenic composition according to the invention (such as the ones ofsection 2 above) with said second immunogenic composition isadministered at the fourth administration.

In another embodiment of said 4-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) withsaid second immunogenic composition is administered at the firstadministration, the second immunogenic composition (such as the ones ofsection 3 above) is administered at the second administration, theconcomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) withsaid second immunogenic composition is administered at the thirdadministration and the second immunogenic composition is administered atthe fourth administration.

In another embodiment of said 4-administration schedule, the secondimmunogenic composition (such as the ones of section 3 above) isadministered at the first administration, the concomitant administrationof the first immunogenic composition according to the invention (such asthe ones of section 2 above) with said second immunogenic composition isadministered at the second administration and the second immunogeniccomposition (such as the ones of section 3 above) is administered at thethird and fourth administrations.

In another embodiment of said 4-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) withsaid second immunogenic composition is administered at the firstadministration, the second immunogenic composition (such as the ones ofsection 3 above) is administered at the second administration and theconcomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) withsaid second immunogenic composition is administered at the third andfourth administrations.

In another embodiment of said 4-administrations schedule, the secondimmunogenic composition (such as the ones of section 3 above) isadministered at the first administration, the concomitant administrationof the first immunogenic composition according to the invention (such asthe ones of section 2 above) with said second immunogenic composition isadministered at the second and third administrations and the secondimmunogenic composition (such as the ones of section 3 above) isadministered at the fourth administration.

In another embodiment of said 4-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) withsaid second immunogenic composition is administered at the firstadministration, the second immunogenic composition (such as the ones ofsection 3 above) is administered at the second and third administrationsand the concomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) withsaid second immunogenic composition is administered at the fourthadministration.

Therefore in an embodiment of said 4-administrations schedule, thesecond immunogenic composition (such as the ones of section 3 above)(designated 2^(nd) IC in the below table) and the concomitantadministration of the first immunogenic composition according to theinvention (such as the ones of section 2 above) with said secondimmunogenic composition (designated 1^(st) IC/2^(nd) IC in the belowtable C) are administered in the following order:

TABLE C Dose Schedule number 1 2 3 4 1 2^(nd) IC 2^(nd) IC 2^(nd) IC 1stIC/2nd IC 2 1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC 3 2^(nd)IC 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 4 1st IC/2nd IC 1st IC/2nd IC2^(nd) IC 2^(nd) IC 5 2^(nd) IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 6 1stIC/2nd IC 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 7 2^(nd) IC 1st IC/2ndIC 1st IC/2nd IC 1st IC/2nd IC 8 1st IC/2nd IC 2^(nd) IC 2^(nd) IC2^(nd) IC 9 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 10 1stIC/2nd IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 11 2^(nd) IC 1st IC/2nd IC2^(nd) IC 2^(nd) IC 12 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 1st IC/2ndIC 13 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC 14 1st IC/2nd IC2^(nd) IC 2^(nd) IC 1st IC/2nd IC

The above table provides the order of administration of the secondimmunogenic composition (such as the ones of section 3 above)(designated 2^(nd) IC in the below table) and the concomitantadministration of the first immunogenic composition according to theinvention (such as the ones of section 2 above) with said secondimmunogenic composition (designated 1^(st) IC/2^(nd) IC in the belowtable) for the different doses, for example schedule number 1 is to beread as: in embodiment of said 3-administrations schedule, the secondimmunogenic composition is administered as the first, second and thirddoses and the concomitant administration of the first immunogeniccomposition according to the invention with said second immunogeniccomposition is administered as the fourth dose.

In a preferred embodiment, the order of administration is according toschedule 1, 3 or 5.

In an embodiment, in any of the 4-administrations schedules disclosedabove the concomitant administration(s) is/are replaced by a concurrentadministration.

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 5 administrations.

In an embodiment, said schedule consists of a series of 5administrations separated by an interval of about 1 month to about 12months. In a particular embodiment, said schedule consists of a seriesof 5 administrations wherein each administration is separated by aninterval of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In aparticular embodiment, said schedule consists of a series of 5administrations wherein each administration is separated by an intervalof about 1 month to about 6 months. In a particular embodiment, saidschedule consists of a series of 5 administrations wherein eachadministration is separated by an interval of about 1, 2, 3, 4, 5 or 6months. In an embodiment, said schedule consists of a series of 5administrations separated by an interval of about 1 month to about 2months. In another embodiment, said schedule consists of a series of 5administrations wherein each administration is separated by an intervalof about 1 month, or a series of 5 administrations wherein eachadministration is separated by an interval of about 2 months.

In an embodiment said schedule consists of a series of 4 administrationswherein each dose is separated by an interval of about 1 month to about3 months followed by a fifth administration about 10 months to about 13months after the first administration. In another embodiment, saidschedule consists of a series of 4 administrations wherein eachadministration is separated by an interval of about 1 month to about 2months followed by a fifth administration about 10 months to about 13months after the first dose. In another embodiment, said scheduleconsists of a series of 4 administrations wherein each dose is separatedby an interval of about 1 month followed by a fifth administration about10 months to about 13 months after the first administration, or a seriesof 4 administrations wherein each administration is separated by aninterval of about 2 months followed by a fifth administration about 10months to about 13 months after the first administration.

In an embodiment of said 5-administrations schedule, the first, second,third and fourth administrations are administered in the first year ofage and the fifth administration is a toddler dose. In an embodiment,said 5-administrations schedule consists of a series of 4administrations wherein each administration is separated by an intervalof about 1 month to about 2 months (for example 28-56 days betweendoses), starting at 2 months of age, and followed by a toddleradministration at 12-18 months of age. In an embodiment, said scheduleconsists of a series of 4 administrations wherein each administrationsis separated by an interval of about 1 month to about 2 months (forexample 28-56 days between doses), starting at 2 months of age, andfollowed by a toddler administration at 12-15 months of age.

In an embodiment of said 5-administrations schedule, the secondimmunogenic composition (such as the ones of section 3 above)(designated 2^(nd) IC in the below table) and the concomitantadministration of the first immunogenic composition according to theinvention (such as the ones of section 2 above) with said secondimmunogenic composition (designated 1^(st) IC/2^(nd) IC in the belowtable D) are administered in the following order:

TABLE D Schedule Dose number 1 2 3 4 5 1 1st IC/2nd IC 1st IC/2nd IC 1stIC/2nd IC 1st IC/2nd IC 2^(nd) IC 2 1st IC/2nd IC 1st IC/2nd IC 1stIC/2nd IC 2^(nd) IC 1st IC/2nd IC 3 1st IC/2nd IC 1st IC/2nd IC 1stIC/2nd IC 2^(nd) IC 2^(nd) IC 4 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC1st IC/2nd IC 1st IC/2nd IC 5 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC2^(nd) IC 1st IC/2nd IC 6 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC 2^(nd)IC 2^(nd) IC 7 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC2^(nd) IC 8 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 1stIC/2nd IC 9 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 2^(nd)IC 10 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 111st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 2^(nd) IC 12 1st IC/2ndIC 2^(nd) IC 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 13 1st IC/2nd IC2^(nd) IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 14 1st IC/2nd IC 2^(nd) IC2^(nd) IC 2^(nd) IC 1st IC/2nd IC 15 1st IC/2nd IC 2^(nd) IC 2^(nd) IC2^(nd) IC 2^(nd) IC 16 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 1st IC/2ndIC 1st IC/2nd IC 17 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC2^(nd) IC 18 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC 1st IC/2ndIC 19 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC 2^(nd) IC 202^(nd) IC 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 21 2^(nd)IC 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 22 2^(nd) IC 1stIC/2nd IC 2^(nd) IC 2^(nd) IC 1st IC/2nd IC 23 2^(nd) IC 1st IC/2nd IC2^(nd) IC 2^(nd) IC 2^(nd) IC 24 2^(nd) IC 2^(nd) IC 1st IC/2nd IC 1stIC/2nd IC 1st IC/2nd IC 25 2^(nd) IC 2^(nd) IC 1st IC/2nd IC 1st IC/2ndIC 2^(nd) IC 26 2^(nd) IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 1st IC/2ndIC 27 2^(nd) IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 2^(nd) IC 28 2^(nd) IC2^(nd) IC 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 29 2^(nd) IC 2^(nd) IC2^(nd) IC 1st IC/2nd IC 2^(nd) IC 30 2^(nd) IC 2^(nd) IC 2^(nd) IC2^(nd) IC 1st IC/2nd IC

The above table provides the order of administration of the secondimmunogenic composition (such as the ones of section 3 above)(designated 2^(nd) IC in the below table) and the concomitantadministration of the first immunogenic composition according to theinvention (such as the ones of section 2 above) with said secondimmunogenic composition (designated 1^(st) IC/2^(nd) IC in the belowtable) for the different doses, for example schedule number 1 is to beread as: in embodiment of said 5-administrations schedule, theconcomitant administration of the first immunogenic compositionaccording to the invention with said second immunogenic composition isadministered as the first, second, third and fourth doses and the secondimmunogenic composition is administered as the fifth dose.

In a preferred embodiment, the order of administration is according toschedule 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28 or 30.

In an embodiment, in any of the 5-administrations schedules disclosedabove the concomitant administration(s) is/are replaced by a concurrentadministration.

In an embodiment, the schedule of vaccination of said sequential doseconsists of a series of 6 administrations.

In an embodiment, said schedule consists of a series of 6administrations separated by an interval of about 1 month to about 12months. In a particular embodiment, said schedule consists of a seriesof 6 administrations wherein each administration is separated by aninterval of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In aparticular embodiment, said schedule consists of a series of 6administrations wherein each administration is separated by an intervalof about 1 month to about 6 months. In a particular embodiment, saidschedule consists of a series of 6 administrations wherein eachadministration is separated by an interval of about 1, 2, 3, 4, 5 or 6months. In an embodiment, said schedule consists of a series of 6administrations separated by an interval of about 1 month to about 2months. In another embodiment, said schedule consists of a series of 6administrations wherein each administration is separated by an intervalof about 1 month, or a series of 6 administrations wherein eachadministration is separated by an interval of about 2 months.

In an embodiment said 6-administrations schedule consists of a series of5 administrations wherein each administration is separated by aninterval of about 1 month to about 2 months followed by a sixthadministration about 10 months to about 13 months after the firstadministration. In another embodiment, said schedule consists of aseries of 5 administrations wherein each administration is separated byan interval of about 1 month followed by a sixth administration about 10months to about 13 months after the first administration, or a series of5 administrations wherein each administration is separated by aninterval of about 2 months followed by a sixth administration about 10months to about 13 months after the first administration.

In an embodiment of said 6-administrations schedule, the first, second,third, fourth and fifth administrations are administered in the firstyear of age and the sixth administration is a toddler administration. Inan embodiment, said 6-administrations schedule consists of a series of 5administrations wherein each administration is separated by an intervalof about 1 month to about 2 months (for example 28-56 days betweenadministrations), starting at 2 months of age, and followed by a toddleradministration at 12-18 months of age. In an embodiment, said scheduleconsists of a series of 5 administrations wherein each administration isseparated by an interval of about 1 month to about 2 months (for example28-56 days between administrations), starting at 2 months of age, andfollowed by a toddler administration at 12-15 months of age.

In an embodiment of said 6-administrations schedule, the secondimmunogenic composition (such as the ones of section 3 above) and theconcomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) withsaid second immunogenic composition are administered in the orderaccording to the any of the 30 schedules provided for the5-administrations schedule (see above table, schedules 1 to 30),followed by a sixth administration. In an embodiment, the secondimmunogenic composition is administered at the sixth administration. Inanother embodiment, the concomitant administration of the firstimmunogenic composition according to the invention with said secondimmunogenic composition is administered at the sixth administration.

In an embodiment, in any of the 6-administrations schedules disclosedabove the concomitant administration(s) is/are replaced by a concurrentadministration.

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 7 administrations.

In an embodiment, said schedule consists of a series of 7administrations separated by an interval of about 1 month to about 12months. In a particular embodiment, said schedule consists of a seriesof 7 administrations wherein each administration is separated by aninterval of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In aparticular embodiment, said schedule consists of a series of 7administrations wherein each administration is separated by an intervalof about 1 month to about 6 months. In a particular embodiment, saidschedule consists of a series of 7 administrations wherein eachadministration is separated by an interval of about 1, 2, 3, 4, 5 or 6months. In an embodiment, said schedule consists of a series of 7administrations separated by an interval of about 1 month to about 2months. In another embodiment, said schedule consists of a series of 7administrations wherein each administration is separated by an intervalof about 1 month, or a series of 7 administrations wherein eachadministration is separated by an interval of about 2 months.

In an embodiment said 7-administrations schedule consists of a series of6 administrations wherein each administration is separated by aninterval of about 1 month followed by a seventh administration about 10months to about 13 months after the first administration.

In an embodiment of said 7-administrations schedule, the first, second,third, fourth, fifth and sixth administrations are administered in thefirst year of age and the seventh administration is a toddleradministration. In an embodiment, said 7-administrations scheduleconsists of a series of 6 administrations wherein each administration isseparated by an interval of about 1 month (for example 28-40 daysbetween administrations), starting at 2 months of age, and followed by atoddler administration at 12-18 months of age. In an embodiment, saidschedule consists of a series of 6 administrations wherein eachadministration is separated by an interval of about 1 month (for example28-40 days between administrations), starting at 2 months of age, andfollowed by a toddler administration at 12-15 months of age.

In an embodiment of said 7-administrations schedule, the secondimmunogenic composition (such as the ones of section 3 above) and theconcomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) withsaid second immunogenic composition are administered in the orderaccording to the any of the schedules provided for the 6-administrationsschedule (see above), followed by a seventh administration. In anembodiment, the second immunogenic composition is administered at theseventh administration. In another embodiment, the concomitantadministration of the first immunogenic composition according to theinvention with said second immunogenic composition is administered atthe seventh administration.

In an embodiment, in any of the 7-administrations schedules disclosedabove the concomitant administration(s) is/are replaced by a concurrentadministration.

In an embodiment, the schedule of vaccination of said sequentialadministration consists of a series of 8 administrations.

In an embodiment, said schedule consists of a series of 8administrations separated by an interval of about 1 month to about 12months. In a particular embodiment, said schedule consists of a seriesof 8 administrations wherein each administration is separated by aninterval of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In aparticular embodiment, said schedule consists of a series of 8administrations wherein each administration is separated by an intervalof about 1 month to about 6 months. In a particular embodiment, saidschedule consists of a series of 8 administrations wherein eachadministration is separated by an interval of about 1, 2, 3, 4, 5 or 6months. In an embodiment, said schedule consists of a series of 8administrations separated by an interval of about 1 month to about 2months. In another embodiment, said schedule consists of a series of 8administrations wherein each administration is separated by an intervalof about 1 month, or a series of 8 administrations wherein eachadministration is separated by an interval of about 2 months.

In an embodiment said 8-administration schedule consists of a series of7 administrations wherein each administration is separated by aninterval of about 1 month followed by an eight administration about 10months to about 13 months after the first administration.

In an embodiment of said 8-administrations schedule, the first, second,third, fourth, fifth, sixth and seventh administrations are administeredin the first year of age and the eighth administration is a toddleradministration. In an embodiment, said 8-administrations scheduleconsists of a series of 7 administrations wherein each administration isseparated by an interval of about 1 month (for example 28-40 daysbetween administrations), starting at 2 months of age, and followed by atoddler administration at 12-18 months of age. In an embodiment, saidschedule consists of a series of 7 administrations wherein eachadministration is separated by an interval of about 1 month (for example28-40 days between administrations), starting at 2 months of age, andfollowed by a toddler administration at 12-15 months of age.

In an embodiment of said 8-administrations schedule, the secondimmunogenic composition (such as the ones of section 3 above) and theconcomitant administration of the first immunogenic compositionaccording to the invention (such as the ones of section 2 above) withsaid second immunogenic composition are administered in the orderaccording to the any of the schedules provided for the 7-administrationsschedule (see above), followed by a eighth dose. In an embodiment, thesecond immunogenic composition is administered at the eighth dose. Inanother embodiment, the concomitant administration of the firstimmunogenic composition according to the invention with said secondimmunogenic composition is administered at the eighth dose.

In an embodiment, in any of the 8-administrations schedules disclosedabove the concomitant administration(s) is/are replaced by a concurrentadministration.

In an embodiment, the immunogenic compositions disclosed herein areadministered by intramuscular or subcutaneous injection.

In an embodiment, the immunogenic compositions are administered byintramuscular injection in a thigh or arm. In an embodiment, theinjection site is the anterolateral thigh muscle or the deltoid muscle.

In an embodiment, the immunogenic compositions are administered bysubcutaneous injection in a thigh or an arm. In an embodiment, theinjection site is the fatty tissue over the anterolateral thigh muscleor the fatty tissue over triceps.

In case of concomitant administration, the first injection can be madein one thigh and the second in the other thigh (preferably in theanterolateral thigh muscles).

Alternatively, the first injection can be made in one arm and the secondin the other arm (preferably in the deltoid muscles). The firstinjection can also be made in a thigh and the second in an arm or thefirst injection in an arm and the second in a thigh.

In an aspect the invention pertains to the kit of the present invention(such as the ones of section 4 above) for use in any of the immunizationschedules disclosed above.

Particular embodiments of the invention are set forth in the followingnumbered paragraphs:

1. An immunogenic composition comprising at least one glycoconjugateselected from the group consisting of a glycoconjugate from S.pneumoniae serotype 15B, a glycoconjugate from S. pneumoniae serotype22F, a glycoconjugate from S. pneumoniae serotype 33F, a glycoconjugatefrom S. pneumoniae serotype 12F, a glycoconjugate from S. pneumoniaeserotype 10A, a glycoconjugate from S. pneumoniae serotype 11A and aglycoconjugate from S. pneumoniae serotype 8, wherein said compositionis a 1, 2, 3, 4, 5, 6 or 7-valent pneumococcal conjugate composition.2. The immunogenic composition of paragraph 1, wherein said compositioncomprises at least one glycoconjugate from S. pneumoniae serotype 15B.3. The immunogenic composition of any one of paragraphs 1-2, whereinsaid composition comprises at least one glycoconjugate from S.pneumoniae serotype 22F.4. The immunogenic composition of any one of paragraphs 1-3, whereinsaid composition comprises at least one glycoconjugate from S.pneumoniae serotype 33F.5. The immunogenic composition of any one of paragraphs 1-4, whereinsaid composition comprises at least one glycoconjugate from S.pneumoniae serotype 12F.6. The immunogenic composition of any one of paragraphs 1-5, whereinsaid composition comprises at least one glycoconjugate from S.pneumoniae serotype 10A.7. The immunogenic composition of any one of paragraphs 1-6, whereinsaid composition comprises at least one glycoconjugate from S.pneumoniae serotype 11A.8. The immunogenic composition of any one of paragraphs 1-7, whereinsaid composition comprises at least one glycoconjugate from S.pneumoniae serotype 8.9. The immunogenic composition of any one of paragraphs 1-8, whereinsaid composition comprises a glycoconjugate from S. pneumoniae serotype15B, a glycoconjugate from S. pneumoniae serotype 22F, glycoconjugatefrom S. pneumoniae serotype 33F, a glycoconjugate from S. pneumoniaeserotype 12F, a glycoconjugate from S. pneumoniae serotype 10A, aglycoconjugate from S. pneumoniae serotype 11A and a glycoconjugate fromS. pneumoniae serotype 8, wherein said composition is a 7-valentpneumococcal conjugate composition.10. The immunogenic composition of any one of paragraphs 1-9, whereinsaid glycoconjugates are individually conjugated to CRM₁₉₇.11. The immunogenic composition of any one of paragraphs 1-9, whereinsaid glycoconjugates are individually conjugated to PD.12. The immunogenic composition of any one of paragraphs 1-9, whereinsaid glycoconjugates are individually conjugated to TT.13. The immunogenic composition of any one of paragraphs 1-9, whereinsaid glycoconjugates are individually conjugated to DT.14. The immunogenic composition of any one of paragraphs 1-13, whereinsaid serotype 15B glycoconjugate has a molecular weight of between 1,000kDa and 20,000 kDa.15. The immunogenic composition of any one of paragraphs 1-14 whereinsaid serotype 15B glycoconjugate has a molecular weight of between10,000 kDa and 16,000 kDa.16. The immunogenic composition of any one of paragraphs 1-15, whereinthe ratio (w/w) of serotype 15B capsular polysaccharide to carrierprotein in serotype 15B glycoconjugate is between 0.5 and 3.17. The immunogenic composition of any one of paragraphs 1-16, whereinthe ratio (w/w) of serotype 15B capsular polysaccharide to carrierprotein in serotype 15B glycoconjugate is between 0.7 and 0.9.18. The immunogenic composition of any one of paragraphs 1-17, whereinsaid serotype 15B glycoconjugate comprises less than about 50% of freeserotype 15B capsular polysaccharide compared to the total amount ofserotype 15B capsular polysaccharide.19. The immunogenic composition of any one of paragraphs 1-18, whereinat least 40% of the serotype 15B glycoconjugates have a K_(d) below orequal to 0.3 in a CL-4B column.20. The immunogenic composition of any one of paragraphs 1-19, whereinsaid serotype 15B glycoconjugate comprises at least 0.1 mM acetate permM serotype 15B capsular polysaccharide.21. The immunogenic composition of any one of paragraphs 1-20, whereinsaid serotype 15B glycoconjugate comprises at least 0.7 mM acetate permM serotype 15B capsular polysaccharide.22. The immunogenic composition of any one of paragraphs 1-21, whereinthe ratio of mM acetate per mM serotype 15B capsular polysaccharide inthe serotype 15B glycoconjugate to mM acetate per mM serotype 15Bcapsular polysaccharide in the isolated polysaccharide is at least 0.6.23. The immunogenic composition of any one of paragraphs 1-22, whereinthe ratio of mM acetate per mM serotype 15B capsular polysaccharide inthe serotype 15B glycoconjugate to mM acetate per mM serotype 15Bcapsular polysaccharide in the activated polysaccharide is at least 0.6.24. The immunogenic composition of any one of paragraphs 1-23, whereinsaid serotype 15B glycoconjugate comprises at least 0.1 mM glycerol permM serotype 15B capsular polysaccharide.25. The immunogenic composition of any one of paragraphs 1-24, whereinsaid serotype 15B glycoconjugate comprises at least 0.5 mM glycerol permM serotype 15B capsular polysaccharide.26. The immunogenic composition of any one of paragraphs 1-25, whereinsaid serotype 15B glycoconjugate comprises at least 0.7 mM glycerol permM serotype 15B capsular polysaccharide.27. The immunogenic composition of any one of paragraphs 1-26, whereinthe degree of conjugation of said serotype 15B glycoconjugate is between2 and 15.28. The immunogenic composition of any one of paragraphs 1-27, whereinsaid serotype 15B glycoconjugate comprise a saccharide having amolecular weight of between 10 kDa and 1,500 kDa.29. The immunogenic composition of any one of paragraphs 1-28, whereinthe carrier protein of said serotype 15B glycoconjugate is CRM₁₉₇.30. The immunogenic composition of any one of paragraphs 1-29, whereinsaid serotype 15B glycoconjugate is prepared using reductive amination.31. The immunogenic composition of any one of paragraphs 1 or 3-30,wherein said serotype 22F glycoconjugate has a molecular weight ofbetween 400 kDa and 15,000 kDa.32. The immunogenic composition of any one of paragraphs 1 or 3-31,wherein said serotype 22F glycoconjugate has a molecular weight ofbetween 1,000 kDa and 8,000 kDa.33. The immunogenic composition of any one of paragraphs 1 or 3-32,wherein the ratio (w/w) of serotype 22F capsular polysaccharide tocarrier protein in serotype 22F glycoconjugate is between 0.5 and 3.34. The immunogenic composition of any one of paragraphs 1 or 3-33,wherein the ratio (w/w) of serotype 22F capsular polysaccharide tocarrier protein in serotype 22F glycoconjugate is between 0.9 and 1.1.35. The immunogenic composition of any one of paragraphs 1 or 3-34,wherein said serotype 22F glycoconjugate comprises less than about 50%of free serotype 22F capsular polysaccharide compared to the totalamount of serotype 22F capsular polysaccharide.36. The immunogenic composition of any one of paragraphs 1 or 3-35,wherein at least 30% of the serotype 22F glycoconjugates have a K_(d)below or equal to 0.3 in a CL-4B column.37. The immunogenic composition of any one of paragraphs 1 or 3-36,wherein said serotype 22F glycoconjugate comprises at least 0.1 mMacetate per mM serotype 22F capsular polysaccharide.38. The immunogenic composition of any one of paragraphs 1 or 3-37,wherein said serotype 22F glycoconjugate comprises at least 0.7 mMacetate per mM serotype 22F capsular polysaccharide.39. The immunogenic composition of any one of paragraphs 1 or 3-38,wherein the ratio of mM acetate per mM serotype 22F capsularpolysaccharide in the serotype 22F glycoconjugate to mM acetate per mMserotype 22F capsular polysaccharide in the isolated polysaccharide isat least 0.6.40. The immunogenic composition of any one of paragraphs 1 or 3-39,wherein the ratio of mM acetate per mM serotype 22F capsularpolysaccharide in the serotype 22F glycoconjugate to mM acetate per mMserotype 22F capsular polysaccharide in the activated polysaccharide isat least 0.6.41. The immunogenic composition of any one of paragraphs 1 or 3-40,wherein the degree of conjugation of said serotype 22F glycoconjugate isbetween 2 and 15.42. The immunogenic composition of any one of paragraphs 1 or 3-41,wherein said serotype 22F glycoconjugate comprise a saccharide having amolecular weight of between 10 kDa and 2,000 kDa.43. The immunogenic composition of any one of paragraphs 1 or 3-42,wherein the carrier protein of said serotype 22F glycoconjugate isCRM₁₉₇.44. The immunogenic composition of any one of paragraphs 1 or 3-43,wherein said serotype 22F glycoconjugate is prepared using reductiveamination.45. The immunogenic composition of any one of paragraphs 1 or 4-44,wherein said serotype 33F glycoconjugate has a molecular weight ofbetween 50 kDa and 20,000 kDa.46. The immunogenic composition of any one of paragraphs 1 or 4-45,wherein said serotype 33F glycoconjugate has a molecular weight ofbetween 1,000 kDa and 5,000 kDa.47. The immunogenic composition of any one of paragraphs 1 or 4-46,wherein the ratio (w/w) of serotype 33F capsular polysaccharide tocarrier protein in serotype 33F glycoconjugate is between 0.2 and 4.48. The immunogenic composition of any one of paragraphs 1 or 4-47,wherein the ratio (w/w) of serotype 33F capsular polysaccharide tocarrier protein in serotype 33F glycoconjugate is between 0.4 and 1.7.49. The immunogenic composition of any one of paragraphs 1 or 4-48,wherein said serotype 33F glycoconjugate comprises less than about 40%of free serotype 33F capsular polysaccharide compared to the totalamount of serotype 33F capsular polysaccharide.50. The immunogenic composition of any one of paragraphs 1 or 4-49,wherein at least 35% of the serotype 33F glycoconjugates have a K_(d)below or equal to 0.3 in a CL-4B column.51. The immunogenic composition of any one of paragraphs 1 or 4-50,wherein said serotype 33F glycoconjugate comprises at least 0.1 mMacetate per mM serotype 33F capsular polysaccharide.52. The immunogenic composition of any one of paragraphs 1 or 4-51,wherein said serotype 33F glycoconjugate comprises at least 0.7 mMacetate per mM serotype 33F capsular polysaccharide.53. The immunogenic composition of any one of paragraphs 1 or 4-52,wherein the ratio of mM acetate per mM serotype 33F capsularpolysaccharide in the serotype 33F glycoconjugate to mM acetate per mMserotype 33F capsular polysaccharide in the isolated polysaccharide isat least 0.6.54. The immunogenic composition of any one of paragraphs 1 or 4-53,wherein the ratio of mM acetate per mM serotype 33F capsularpolysaccharide in the serotype 33F glycoconjugate to mM acetate per mMserotype 33F capsular polysaccharide in the activated polysaccharide isat least 0.6.55. The immunogenic composition of any one of paragraphs 1 or 4-54,wherein the degree of conjugation of said serotype 33F glycoconjugate isbetween 2 and 20.56. The immunogenic composition of any one of paragraphs 1 or 4-55,wherein said serotype 33F glycoconjugate comprise a saccharide having amolecular weight of between 10 kDa and 2,000 kDa.57. The immunogenic composition of any one of paragraphs 1 or 4-56,wherein said serotype 33F glycoconjugate comprise at least one covalentlinkage between the carrier protein and saccharide for every 2 to 25saccharide repeat units.58. The immunogenic composition of any one of paragraphs 1 or 4-57,wherein the carrier protein of said serotype 33F glycoconjugate isCRM₁₉₇.59. The immunogenic composition of any one of paragraphs 1 or 4-58,wherein said serotype 33F glycoconjugate is prepared using reductiveamination.60. The immunogenic composition of any one of paragraphs 1 or 4-59,wherein said serotype 33F glycoconjugate is prepared using eTECconjugation.61. The immunogenic composition of paragraph 60, wherein said serotype33F glycoconjugate is represented by the general formula (III):

where the atoms that comprise the eTEC spacer are contained in thecentral box.62. The immunogenic composition of any one of paragraphs 1 or 5-61,wherein said serotype 12F glycoconjugate has a molecular weight ofbetween 50 kDa and 20,000 kDa.63. The immunogenic composition of any one of paragraphs 1 or 5-62,wherein said serotype 12F glycoconjugate has a molecular weight ofbetween 500 kDa and 5,000 kDa.64. The immunogenic composition of any one of paragraphs 1 or 5-63,wherein the ratio (w/w) of serotype 12F capsular polysaccharide tocarrier protein in serotype 12F glycoconjugate is between 0.2 and 4.65. The immunogenic composition of any one of paragraphs 1 or 5-64,wherein the ratio (w/w) of serotype 12F capsular polysaccharide tocarrier protein in serotype 12F glycoconjugate is between 0.8 and 1.8.66. The immunogenic composition of any one of paragraphs 1 or 5-65,wherein said serotype 22F glycoconjugate comprises less than about 50%of free serotype 12F capsular polysaccharide compared to the totalamount of serotype 12F capsular polysaccharide.67. The immunogenic composition of any one of paragraphs 1 or 5-66,wherein at least 35% of the serotype 12F glycoconjugates have a K_(d)below or equal to 0.3 in a CL-4B column.68. The immunogenic composition of any of one of paragraphs 1 or 5-67,wherein the degree of conjugation of said serotype 12F glycoconjugate isbetween 2 and 20.69. The immunogenic composition of any one of paragraphs 1 or 5-68,wherein said serotype 12F glycoconjugate comprise a saccharide having amolecular weight of between 10 kDa and 2,000 kDa.70. The immunogenic composition of any one of paragraphs 1 or 5-69,wherein said serotype 12F glycoconjugate comprise at least one covalentlinkage between the carrier protein and saccharide for every 2 to 25saccharide repeat units.71. The immunogenic composition of any one of paragraphs 1 or 5-70,wherein the carrier protein of said serotype 12F glycoconjugate isCRM₁₉₇.72. The immunogenic composition of any one of paragraphs 1 or 5-71,wherein said serotype 12F glycoconjugate is prepared using reductiveamination.73. The immunogenic composition of any one of paragraphs 1 or 5-72,wherein said serotype 12F glycoconjugate is prepared usingTEMPO/NCS-reductive amination.74. The immunogenic composition of any one of paragraphs 1 or 6-73,wherein said serotype 10A glycoconjugate has a molecular weight ofbetween 50 kDa and 20,000 kDa.75. The immunogenic composition of any one of paragraphs 1 or 6-74,wherein said serotype 10A glycoconjugate has a molecular weight ofbetween 1,000 kDa and 10,000 kDa.76. The immunogenic composition of any one of paragraphs 1 or 6-75,wherein the ratio (w/w) of serotype 10A capsular polysaccharide tocarrier protein in serotype 10A glycoconjugate is between 0.5 and 3.77. The immunogenic composition of any one of paragraphs 1 or 6-76,wherein the ratio (w/w) of serotype 10A capsular polysaccharide tocarrier protein in serotype 10A glycoconjugate is between 0.8 and 1.2.78. The immunogenic composition of any one of paragraphs 1 or 6-77,wherein said serotype 10A glycoconjugate comprises less than about 50%of free serotype 10A capsular polysaccharide compared to the totalamount of serotype 10A capsular polysaccharide.79. The immunogenic composition of any one of paragraphs 1 or 6-78,wherein at least 30% of the serotype 10A glycoconjugates have a K_(d)below or equal to 0.3 in a CL-4B column.80. The immunogenic composition of any of one of paragraphs 1 or 6-79,wherein the degree of conjugation of said serotype 10A glycoconjugate isbetween 2 and 15.81. The immunogenic composition of any one of paragraphs 1 or 6-80,wherein said serotype 10A glycoconjugate comprise a saccharide having amolecular weight of between 10 kDa and 2,000 kDa.82. The immunogenic composition of any one of paragraphs 1 or 6-81,wherein the carrier protein of said serotype 10A glycoconjugate isCRM₁₉₇.83. The immunogenic composition of any one of paragraphs 1 or 6-82,wherein said serotype 10A glycoconjugate is prepared using reductiveamination.84. The immunogenic composition of any one of paragraphs 1 or 7-83,wherein said serotype 11A glycoconjugate has a molecular weight ofbetween 50 kDa and 20,000 kDa.85. The immunogenic composition of any one of paragraphs 1 or 7-84,wherein said serotype 11A glycoconjugate has a molecular weight ofbetween 500 kDa and 20,000 kDa.86. The immunogenic composition of any of one of paragraphs 1 or 7-85,wherein the ratio (w/w) of serotype 11A capsular polysaccharide tocarrier protein in serotype 11A glycoconjugate is between 0.2 and 4.87. The immunogenic composition of any one of paragraphs 1 or 7-86,wherein the ratio (w/w) of serotype 11A capsular polysaccharide tocarrier protein in serotype 11A glycoconjugate is between 0.8 and 1.6.88. The immunogenic composition of any one of paragraphs 1 or 7-87,wherein said serotype 11A glycoconjugate comprises less than about 50%of free serotype 11A capsular polysaccharide compared to the totalamount of serotype 11A capsular polysaccharide.89. The immunogenic composition of any one of paragraphs 1 or 7-88,wherein at least 30% of the serotype 11A glycoconjugates have a K_(d)below or equal to 0.3 in a CL-4B column.90. The immunogenic composition of any one of paragraphs 1 or 7-89,wherein said serotype 11A glycoconjugate comprises at least 0.3 mMacetate per mM serotype 11A capsular polysaccharide.91. The immunogenic composition of any one of paragraphs 1 or 7-90,wherein said serotype 11A glycoconjugate comprises at least 1.8 mMacetate per mM serotype 11A capsular polysaccharide.92. The immunogenic composition of any one of paragraphs 1 or 7-91,wherein the ratio of mM acetate per mM serotype 11A capsularpolysaccharide in the serotype 11A glycoconjugate to mM acetate per mMserotype 11A capsular polysaccharide in the isolated polysaccharide isat least 0.6.93. The immunogenic composition of any one of paragraphs 1 or 7-92,wherein the ratio of mM acetate per mM serotype 11A capsularpolysaccharide in the serotype 11A glycoconjugate to mM acetate per mMserotype 11A capsular polysaccharide in the activated polysaccharide isat least 0.694. The immunogenic composition of any one of paragraphs 1 or 7-93,wherein said serotype 11A glycoconjugate comprises at least 0.1 mMglycerol per mM serotype 11A capsular polysaccharide.95. The immunogenic composition of any one of paragraphs 1 or 7-94,wherein said serotype 11A glycoconjugate comprises at least 0.4 mMglycerol per mM serotype 11A capsular polysaccharide.96. The immunogenic composition of any one of paragraphs 1 or 7-95,wherein the degree of conjugation of said serotype 11A glycoconjugate isbetween 1 and 15.97. The immunogenic composition of any one of paragraphs 1 or 7-96,wherein said serotype 11A glycoconjugate comprise a saccharide having amolecular weight of between 10 kDa and 2,000 kDa.98. The immunogenic composition of any one of paragraphs 1 or 7-97,wherein the carrier protein of said serotype 11A glycoconjugate isCRM₁₉₇.99. The immunogenic composition of any one of paragraphs 1 or 7-98,wherein said serotype 11A glycoconjugate is prepared using reductiveamination.100. The immunogenic composition of any one of paragraphs 1 or 8-99,wherein said serotype 8 glycoconjugate has a molecular weight of between50 kDa and 20,000 kDa.101. The immunogenic composition of any one of paragraphs 1 or 8-100,wherein said serotype 8 glycoconjugate has a molecular weight of between1,000 kDa and 15,000 kDa.102. The immunogenic composition of any one of paragraphs 1 or 8-101,wherein the ratio (w/w) of serotype 8 capsular polysaccharide to carrierprotein in serotype 8 glycoconjugate is between 0.2 and 4.103. The immunogenic composition of any one of paragraphs 1 or 8-102,wherein the ratio (w/w) of serotype 8 capsular polysaccharide to carrierprotein in serotype 8 glycoconjugate is between 0.8 and 1.5.104. The immunogenic composition of any one of paragraphs 1 or 8-103,wherein said serotype 8 glycoconjugate comprises less than about 50% offree serotype 8 capsular polysaccharide compared to the total amount ofserotype 8 capsular polysaccharide.105. The immunogenic composition of any one of paragraphs 1 or 8-104,wherein at least 30% of the serotype 8 glycoconjugates have a K_(d)below or equal to 0.3 in a CL-4B column.106. The immunogenic composition of any one of paragraphs 1 or 8-105,wherein the degree of conjugation of said serotype 8 glycoconjugate isbetween 2 and 20.107. The immunogenic composition of any one of paragraphs 1 or 8-106,wherein said serotype 8 glycoconjugate comprise a saccharide having amolecular weight of between 10 kDa and 2,000 kDa.108. The immunogenic composition of any one of paragraphs 1 or 8-107,wherein the carrier protein of said serotype 8 glycoconjugate is CRM₁₉₇.109. The immunogenic composition of any one of paragraphs 1 or 8-108,wherein said serotype 8 glycoconjugate is prepared using reductiveamination.110. The immunogenic composition of any one of paragraphs 1-109, whereineach dose of said immunogenic composition comprises 0.1 μg to 100 μg ofpolysaccharide of each serotype.111. The immunogenic composition of any one of paragraphs 1-110, whereineach dose of said immunogenic composition comprises 1.0 μg to 10 μg ofpolysaccharide of each serotype.112. The immunogenic composition of any one of paragraphs 1-111, whereineach dose of said immunogenic composition comprises about 1.0 μg, about1.2 μg, about 1.4 μg, about 1.6 μg, about 1.8 μg, 2.0 μg, about 2.2 μg,about 2.4 μg, about 2.6 μg, about 2.8 μg, about 3.0 μg, about 3.2 μg,about 3.4 μg, about 3.6 μg, about 3.8 μg, about 4.0 μg, about 4.2 μg,about 4.4 μg, about 4.6 μg, about 4.8 μg, about 5.0 μg, about 5.2 μg,about 5.4 μg, about 5.6 μg, about 5.8 μg or about 6.0 μg ofpolysaccharide for each serotype glycoconjugate.113. The immunogenic composition of any one of paragraphs 1-112, whereineach dose of said immunogenic composition comprises about 1.5 μg toabout 3.0 μg of polysaccharide for each glycoconjugate from S.pneumoniae serotype 8, 10A, 11A, 12F, 15B, 22F, and/or 33F, if present.114. The immunogenic composition of any one of paragraphs 1-113, whereineach dose of said immunogenic composition comprises 10 μg to 150 μg ofcarrier protein.115. The immunogenic composition of any one of paragraphs 1-114, whereineach dose of said immunogenic composition comprises about 1 μg, about 2μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg,about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg,about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg,about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg,about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg,about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about63 μg, about 64 μg, about 65 μg, about 66 μg, about 67 μg, about 68 μg,about 69 μg, about 70 μg, about 71 μg, about 72 μg, about 73 μg, about74 μg or about 75 μg of carrier protein.116. The immunogenic composition of any one of paragraphs 1-115, whereinsaid immunogenic composition further comprises at least one antigen fromother pathogens.117. The immunogenic composition of any one of paragraphs 1-116, whereinsaid immunogenic composition further comprises at least one antigenselected from the group consisting of a diphtheria toxoid (D), a tetanustoxoid (T), a pertussis antigen (P), an acellular pertussis antigen(Pa), a hepatitis B virus (HBV) surface antigen (HBsAg), a hepatitis Avirus (HAV) antigen, a conjugated Haemophilus influenzae type b capsularsaccharide (Hib), and inactivated poliovirus vaccine (IPV).118. The immunogenic composition of any one of paragraphs 1-117, whereinsaid immunogenic composition further comprises D, T and Pa.119. The immunogenic composition of any one of paragraphs 1-117, whereinsaid immunogenic composition further comprises D, T, Pa and Hib.120. The immunogenic composition of any one of paragraphs 1-117, whereinsaid immunogenic composition further comprises D, T, Pa and IPV.121. The immunogenic composition of any one of paragraphs 1-117, whereinsaid immunogenic composition further comprises D, T, Pa and HBsAg.122. The immunogenic composition of any one of paragraphs 1-117, whereinsaid immunogenic composition further comprises D, T, Pa, HBsAg and IPV.123. The immunogenic composition of any one of paragraphs 1-117, whereinsaid immunogenic composition further comprises D, T, Pa, HBsAg and Hib.124. The immunogenic composition of any one of paragraphs 1-117, whereinsaid immunogenic composition further comprises D, T, Pa, HBsAg, IPV andHib.125. The immunogenic composition of any one of paragraphs 1-124, whereinsaid immunogenic composition further comprises a conjugated N.meningitidis serogroup Y capsular saccharide (MenY).126. The immunogenic composition of any one of paragraphs 1-125, whereinsaid immunogenic composition further comprises a conjugated N.meningitidis serogroup C capsular saccharide (MenC).127. The immunogenic composition of any one of paragraphs 1-126, whereinsaid immunogenic composition further comprises a conjugated N.meningitidis serogroup A capsular saccharide (MenA).128. The immunogenic composition of any one of paragraphs 1-127, whereinsaid immunogenic composition further comprises a conjugated N.meningitidis serogroup W135 capsular saccharide (MenW135).129. The immunogenic composition of any one of paragraphs 1-128, whereinsaid immunogenic composition further comprises a conjugated N.meningitidis serogroup Y capsular saccharide (MenY) and a conjugated N.meningitidis serogroup C capsular saccharide (MenC).130. The immunogenic composition of any one of paragraphs 1-124, whereinsaid immunogenic composition further comprises a conjugated N.meningitidis serogroup W135 capsular saccharide (MenW135), a conjugatedN. meningitidis serogroup Y capsular saccharide (MenY), and/or aconjugated N. meningitidis serogroup C capsular saccharide (MenC).131. The immunogenic composition of any one of paragraphs 1-124, whereinsaid immunogenic composition further comprises a conjugated N.meningitidis serogroup A capsular saccharide (MenA), a conjugated N.meningitidis serogroup W135 capsular saccharide (MenW135), a conjugatedN. meningitidis serogroup Y capsular saccharide (MenY), and/or aconjugated N. meningitidis serogroup C capsular saccharide (MenC).132. The immunogenic composition of any one of paragraphs 1-131, whereinsaid immunogenic composition further comprises at least one adjuvant.133. The immunogenic composition of any one of paragraphs 1-132, whereinsaid immunogenic composition further comprises at least one adjuvantselected from the group consisting of aluminum phosphate, aluminumsulfate or aluminum hydroxide, calcium phosphate, liposomes, anoil-in-water emulsion, MF59 (4.3% w/v squalene, 0.5% w/v polysorbate 80,0.5% w/v sorbitan trioleate), a water-in-oil emulsion, MONTANIDE™,poly(D,L-lactide-co-glycolide) (PLG) microparticles andpoly(D,L-lactide-co-glycolide) (PLG) nanoparticles.134. The immunogenic composition of any one of paragraphs 1-131 whereinsaid immunogenic composition further comprise at least one adjuvantselected from the group consisting of aluminum phosphate, aluminumsulfate and aluminum hydroxide.135. The immunogenic composition of any one of paragraphs 1-131 whereinsaid immunogenic composition further comprise aluminum phosphate asadjuvant.136. The immunogenic composition of any one of paragraphs 1-131 whereinsaid immunogenic composition further comprise aluminum sulfate asadjuvant.137. The immunogenic composition of any one of paragraphs 1-131 whereinsaid immunogenic composition further comprise aluminum hydroxide asadjuvant.138. The immunogenic composition of any one of paragraphs 1-131 whereinsaid immunogenic composition comprise from 0.1 mg/mL to 1 mg/mL ofelemental aluminum in the form of aluminum phosphate as adjuvant.139. The immunogenic composition of any one of paragraphs 1-131 whereinsaid immunogenic composition comprise from 0.2 mg/mL to 0.3 mg/mL ofelemental aluminum in the form of aluminum phosphate as adjuvant.140. The immunogenic composition of any one of paragraphs 1-131 whereinsaid immunogenic composition comprise about 0.25 mg/mL of elementalaluminum in the form of aluminum phosphate as adjuvant.141. The immunogenic composition of any one of paragraphs 1-140, whereinsaid immunogenic composition further comprises a CpG Oligonucleotide.142. The immunogenic composition of any one of paragraphs 1-141, whereinsaid immunogenic composition is formulated in a liquid form.143. The immunogenic composition of any one of paragraphs 1-141, whereinsaid immunogenic composition is formulated in a lyophilized form.144. The immunogenic composition of any one of paragraphs 1-142, whereinsaid immunogenic composition is formulated in an aqueous liquid form.145. The immunogenic composition of any one of paragraphs 1-144, whereinsaid immunogenic composition comprises one or more of a buffer, a salt,a divalent cation, a non-ionic detergent, a cryoprotectant such as asugar, and an anti-oxidant such as a free radical scavenger or chelatingagent, or any combinations thereof.146. The immunogenic composition of any one of paragraphs 1-145, whereinsaid immunogenic composition comprises a buffer.147. The immunogenic composition of paragraph 146, wherein said bufferhas a pKa of about 3.5 to about 7.5.148. The immunogenic composition of any one of paragraphs 146-147,wherein said buffer is phosphate, succinate, histidine or citrate.149. The immunogenic composition of any one of paragraphs 146-148,wherein said buffer is succinate at a final concentration of 1.0 mM to10 mM.150. The immunogenic composition of any one of paragraphs 146-149,wherein said buffer is succinate at a final concentration of about 5.0mM.151. The immunogenic composition of any one of paragraphs 1-150, whereinthe immunogenic composition comprises a salt.152. The immunogenic composition of paragraph 151, wherein said salt isselected from the group consisting of magnesium chloride, potassiumchloride, sodium chloride and a combination thereof.153. The immunogenic composition of any one of paragraphs 151-152,wherein said salt is sodium chloride.154. The immunogenic composition of any one of paragraphs 151-153,wherein said salt is sodium chloride at a concentration of about 150 mM.155. The immunogenic composition of any one of paragraphs 1-154, whereinthe immunogenic composition comprises a surfactant.156. The immunogenic composition of paragraph 155, wherein saidsurfactant is selected from the group consisting of polysorbate 20,polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80,polysorbate 85, Triton N-101, Triton X-100, oxtoxynol 40, nonoxynol-9,triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660hydroxystearate, polyoxyethylene-35-ricinoleate, soy lecithin and apoloxamer.157. The immunogenic composition of any one of paragraphs 155-156,wherein said surfactant is selected from the group polysorbate 20,polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80,polysorbate 85 and a poloxamer.158. The immunogenic composition of any one of paragraphs 155-157,wherein said surfactant is polysorbate 80.159. The immunogenic composition of any one of paragraphs 155-158,wherein the surfactant is polysorbate 80 at a final concentration of atleast 0.0001% to 10% weight to weight (w/w).160. The immunogenic composition of any one of paragraphs 155-159,wherein the surfactant is polysorbate 80 at a final concentration of atleast 0.001% to 1% weight to weight (w/w).161. The immunogenic composition of any one of paragraphs 155-160,wherein the surfactant is polysorbate 80 at a final concentration of atleast 0.01% to 1% weight to weight (w/w).162. The immunogenic composition of any one of paragraphs 155-161,wherein the surfactant is polysorbate 80 at a final concentration of0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1%weight to weight (w/w).163. The immunogenic composition of any one of paragraphs 1-162, whereinsaid immunogenic composition has a pH of 5.5 to 7.5.164. The immunogenic composition of any one of paragraphs 1-163, whereinsaid immunogenic composition has a pH of 5.6 to 7.0.165. The immunogenic composition of any one of paragraphs 1-164, whereinsaid immunogenic composition has a pH of 5.8 to 6.0.166. A kit comprising: (a) a first immunogenic composition comprisingsaid immunogenic composition of any one of paragraphs 1-165; and (b) asecond immunogenic composition comprising at least one glycoconjugatefrom a Streptococcus pneumoniae serotype selected from the groupconsisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F,23F, 22F and 33F.167. The kit of paragraph 166, wherein said second immunogeniccomposition comprises glycoconjugates from S. pneumoniae serotypes 4,6B, 9V, 14, 18C, 19F and 23F.168. The kit of paragraph 166, wherein said second immunogeniccomposition comprises glycoconjugates from S. pneumoniae serotypes 1, 4,5, 6B, 7F, 9V, 14, 18C, 19F and 23F.169. The kit of paragraph 166, wherein said second immunogeniccomposition comprises glycoconjugates from S. pneumoniae serotypes 1, 4,5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.170. The kit of paragraph 166, wherein said second immunogeniccomposition comprises glycoconjugates from S. pneumoniae serotypes 1, 3,4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.171. The kit of paragraph 166, wherein said second immunogeniccomposition comprises glycoconjugates from S. pneumoniae serotypes 1, 4,5, 6B, 7F, 9V, 14, 18C, 19F, 23F and 22F.172. The kit of paragraph 166, wherein said second immunogeniccomposition comprises glycoconjugates from S. pneumoniae serotypes 1, 4,5, 6B, 7F, 9V, 14, 18C, 19F, 23F and 33F.173. The kit of paragraph 166, wherein said second immunogeniccomposition comprises glycoconjugates from S. pneumoniae serotypes 1, 4,5, 6B, 7F, 9V, 14, 18C, 19F, 23F, 22F and 33F.174. The kit of paragraph 166, wherein said second immunogeniccomposition comprises glycoconjugates from S. pneumoniae serotypes 1, 3,4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 22F.175. The kit of paragraph 166, wherein said second immunogeniccomposition comprises glycoconjugates from S. pneumoniae serotypes 1, 3,4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 33F.176. 175. The kit of paragraph 166, wherein said second immunogeniccomposition comprises glycoconjugates from S. pneumoniae serotypes 1, 3,4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 22F and 33F.177. The kit of any one of paragraphs 166-176, wherein saidglycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and23F are conjugated to CRM₁₉₇.178. The kit of any one of paragraphs 166-177, wherein saidglycoconjugates from S. pneumoniae serotypes 1, 5 and 7F are conjugatedto CRM₁₉₇.179. The kit of any one of paragraphs 166-178, wherein saidglycoconjugates from S. pneumoniae serotypes 6A and 19A are conjugatedto CRM₁₉₇.180. The kit of any one of paragraphs 166-179, wherein saidglycoconjugate from S. pneumoniae serotypes 3 is conjugated to CRM₁₉₇.181. The kit of any one of paragraphs 166-180, wherein saidglycoconjugate from S. pneumoniae serotypes 22F is conjugated to CRM₁₉₇.182. The kit of any one of paragraphs 166-181, wherein saidglycoconjugate from S. pneumoniae serotypes 33F is conjugated to CRM₁₉₇.183. The kit of any one of paragraphs 166-182, wherein saidglycoconjugates are all individually conjugated to CRM₁₉₇.184. The kit of any one of paragraphs 166-176, wherein saidglycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and23F are individually conjugated to PD.185. The kit of any one of paragraphs 166-176 or 184, wherein saidglycoconjugate from S. pneumoniae serotype 18C is conjugated to TT.186. The kit of any one of paragraphs 166-176 or 184-185, wherein saidglycoconjugate from S. pneumoniae serotype 19F is conjugated to DT.187. The kit of any one of paragraphs 166-176 or 184-186, wherein saidglycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14and/or 23F are individually conjugated to PD, said glycoconjugate fromS. pneumoniae serotype 18C is conjugated to TT and said glycoconjugatefrom S. pneumoniae serotype 19F is conjugated to DT.188. The kit of any one of paragraphs 184-187, wherein saidglycoconjugate from S. pneumoniae serotypes 22F is conjugated to CRM₁₉₇.189. The kit of any one of paragraphs 184-188, wherein saidglycoconjugate from S. pneumoniae serotypes 33F is conjugated to CRM₁₉₇190. The kit of any one of paragraphs 166-189, wherein said secondimmunogenic composition is a 7, 8, 9, 10, 11, 12, 13, 14 or 15-valentpneumococcal conjugate composition.191. The kit of any one of paragraphs 166-190, wherein said secondimmunogenic composition is a 10, 11, 12, 13, 14 or 15-valentpneumococcal conjugate composition.192. The kit of any one of paragraphs 166-191, wherein said secondimmunogenic composition is a 13-valent pneumococcal conjugatecomposition.193. The kit of any one of paragraphs 166-191, wherein said secondimmunogenic composition is an 11-valent pneumococcal conjugatecomposition wherein said 11 conjugates consists of glycoconjugates fromS. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F individuallyconjugated to PD, glycoconjugate from S. pneumoniae serotype 18Cconjugated to TT, glycoconjugate from S. pneumoniae serotype 19Fconjugated to DT and glycoconjugate from S. pneumoniae serotype 22Fconjugated to CRM₁₉₇.194. The kit of any one of paragraphs 166-191, wherein said secondimmunogenic composition is an 11-valent pneumococcal conjugatecomposition wherein said 11 conjugates consists of glycoconjugates fromS. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F individuallyconjugated to PD, glycoconjugate from S. pneumoniae serotype 18Cconjugated to TT, glycoconjugate from S. pneumoniae serotype 19Fconjugated to DT and glycoconjugate from S. pneumoniae serotype 33Fconjugated to CRM₁₉₇.195. The kit of any one of paragraphs 166-191, wherein said secondimmunogenic composition is a 12-valent pneumococcal conjugatecomposition wherein said 12 conjugates consists of glycoconjugates fromS. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F individuallyconjugated to PD, glycoconjugate from S. pneumoniae serotype 18Cconjugated to TT, glycoconjugate from S. pneumoniae serotype 19Fconjugated to DT, glycoconjugate from S. pneumoniae serotype 22Fconjugated to CRM₁₉₇ and glycoconjugate from S. pneumoniae serotype 33Fconjugated to CRM₁₉₇.196. The kit of any one of paragraphs 166-192, wherein said secondimmunogenic composition is a 13-valent pneumococcal conjugatecomposition wherein said 13 conjugates consists of glycoconjugates fromS. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19Fand 23F individually conjugated to CRM₁₉₇.197. The kit of any one of paragraphs 166-191, wherein said secondimmunogenic composition is a 14-valent pneumococcal conjugatecomposition wherein said 14 conjugates consists of glycoconjugates fromS. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F,23F and 22F individually conjugated to CRM₁₉₇.198. The kit of any one of paragraphs 166-191, wherein said secondimmunogenic composition is a 14-valent pneumococcal conjugatecomposition wherein said 14 conjugates consists of glycoconjugates fromS. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F,23F and 33F individually conjugated to CRM₁₉₇.199. The kit of any one of paragraphs 166-191, wherein said secondimmunogenic composition is a 15-valent pneumococcal conjugatecomposition wherein said 15 conjugates consists of glycoconjugates fromS. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F,23F, 22F and 33F individually conjugated to CRM₁₉₇.200. The kit of any one of paragraphs 166-199, wherein saidglycoconjugates of the second immunogenic composition are all conjugatedto the carrier protein by reductive amination.201. The kit of any one of paragraphs 166-200, wherein each dose of saidsecond immunogenic composition comprises 1.0 μg to 10 μg ofpolysaccharide of each serotype.202. The kit of any one of paragraphs 166-201, wherein each dose of saidsecond immunogenic composition comprises 10 μg to 150 μg of carrierprotein.203. The kit of any one of paragraphs 166-202, wherein each dose of saidsecond immunogenic composition comprises about 15 μg, about 16 μg, about17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg,about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg,about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg,about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg or about50 μg of carrier protein.204. The kit of any one of paragraphs 166-203, wherein said secondimmunogenic composition further comprises at least one antigen fromother pathogens.205. The kit of any one of paragraphs 166-204, wherein said secondimmunogenic composition further comprises at least one adjuvant.206. The kit of any one of paragraphs 166-204, wherein said secondimmunogenic composition further comprises at least one adjuvant selectedfrom the group consisting of aluminum phosphate, aluminum sulfate andaluminum hydroxide.207. The kit of any one of paragraphs 166-204, wherein said secondimmunogenic composition further comprises aluminum phosphate asadjuvant.208. The kit of any one of paragraphs 166-204, wherein said secondimmunogenic composition further comprises from 0.2 mg/mL to 0.3 mg/mL ofelemental aluminum in the form of aluminum phosphate as adjuvant.209. The kit of any one of paragraphs 166-204, wherein said secondimmunogenic composition further comprises about 0.25 mg/mL of elementalaluminum in the form of aluminum phosphate as adjuvant.210. The kit of any one of paragraphs 166-209, wherein said secondimmunogenic composition further comprises a buffer.211. The kit of paragraph 210, wherein said buffer has a pKa of about3.5 to about 7.5.212. The kit of any one of paragraphs 210-211, wherein said buffer isphosphate, succinate, histidine or citrate.213. The kit of paragraph 212, wherein said buffer is succinate at afinal concentration of about 5.0 mM.214. The kit of any one of paragraphs 166-213, wherein said secondimmunogenic composition further comprises a salt.215. The kit of paragraph 214, wherein said salt is selected from thegroup consisting of magnesium chloride, potassium chloride, sodiumchloride and a combination thereof.216. The kit of any one of paragraphs 166-215, wherein said secondimmunogenic composition comprises sodium chloride at a finalconcentration of 150 mM.217. The kit of any one of paragraphs 166-216, wherein said secondimmunogenic composition further comprises a surfactant.218. The kit of paragraph 217, wherein said surfactant is polysorbate80.219. The kit of paragraph 218, wherein the final concentration ofpolysorbate 80 is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%,0.08%, 0.09% or 0.1% (w/w).220. The kit of any one of paragraphs 166-219, wherein said secondimmunogenic composition has a pH of 5.8 to 6.0.221. The kit of any one of paragraphs 166-220, wherein said firstimmunogenic composition and said second immunogenic composition are inseparate containers.222. The kit of any one of paragraphs 166-221, wherein said first andsecond immunogenic compositions are formulated in a liquid form.223. The kit of any one of paragraphs 166-221, wherein said first andsecond immunogenic compositions are formulated in a lyophilized form.224. The kit of any one of paragraphs 166-221, wherein said firstimmunogenic composition is in a liquid form and said second immunogeniccomposition is in a lyophilized form.225. The kit of any one of paragraphs 166-221, wherein said firstimmunogenic composition is in lyophilized form and said secondimmunogenic composition is in liquid form.226. The immunogenic composition of any one of paragraphs 1-165, whereinsaid immunogenic composition is simultaneously, concurrently,concomitantly or sequentially administered with a second immunogeniccomposition.227. The immunogenic composition of any one of paragraphs 1-165, forsimultaneous, concurrent, concomitant or sequential administration witha second immunogenic composition.228. The immunogenic composition of any one of paragraphs 1-165 forsimultaneous, concurrent, concomitant or sequential administration withany of the immunogenic compositions disclosed at section 3 above.229. The immunogenic composition of any one of paragraphs 226-228,wherein said second immunogenic composition comprises at least oneglycoconjugate from a Streptococcus pneumoniae serotype selected fromthe group consisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C,19A, 19F, 23F, 22F and 33F.230. The immunogenic composition of paragraph 229, wherein said secondimmunogenic composition comprises glycoconjugates from S. pneumoniaeserotypes 4, 6B, 9V, 14, 18C, 19F and 23F.231. The immunogenic composition of paragraph 229, wherein said secondimmunogenic composition comprises glycoconjugates from S. pneumoniaeserotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.232. The immunogenic composition of paragraph 229, wherein said secondimmunogenic composition comprises glycoconjugates from S. pneumoniaeserotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.233. The immunogenic composition of paragraph 229, wherein said secondimmunogenic composition comprises glycoconjugates from S. pneumoniaeserotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.234. The immunogenic composition of paragraph 229, wherein said secondimmunogenic composition comprises glycoconjugates from S. pneumoniaeserotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 23F and 22F.235. The immunogenic composition of paragraph 229, wherein said secondimmunogenic composition comprises glycoconjugates from S. pneumoniaeserotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 23F and 33F.236. The immunogenic composition of paragraph 229, wherein said secondimmunogenic composition comprises glycoconjugates from S. pneumoniaeserotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 23F, 22F and 33F.237. The immunogenic composition of paragraph 229, wherein said secondimmunogenic composition comprises glycoconjugates from S. pneumoniaeserotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 22F.238. The immunogenic composition of paragraph 229, wherein said secondimmunogenic composition comprises glycoconjugates from S. pneumoniaeserotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 33F.239. The immunogenic composition of paragraph 229, wherein said secondimmunogenic composition comprises glycoconjugates from S. pneumoniaeserotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 22F and33F.240. The immunogenic composition of any one of paragraphs 229-239,wherein said glycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14,18C, 19F and 23F are conjugated to CRM₁₉₇.241. The immunogenic composition of any one of paragraphs 229-240,wherein said glycoconjugates from S. pneumoniae serotypes 1, 5 and 7Fare conjugated to CRM₁₉₇.242. The immunogenic composition of any one of paragraphs 229-241,wherein said glycoconjugates from S. pneumoniae serotypes 6A and 19A areconjugated to CRM₁₉₇.243. The immunogenic composition of any one of paragraphs 229-242,wherein said glycoconjugate from S. pneumoniae serotypes 3 is conjugatedto CRM₁₉₇.244. The immunogenic composition of any one of paragraphs 229-243,wherein said glycoconjugate from S. pneumoniae serotypes 22F isconjugated to CRM₁₉₇.245. The immunogenic composition of any one of paragraphs 229-244,wherein said glycoconjugate from S. pneumoniae serotypes 33F isconjugated to CRM₁₉₇.246. The immunogenic composition of any one of paragraphs 229-246,wherein said glycoconjugates are all individually conjugated to CRM₁₉₇.247. The immunogenic composition of any one of paragraphs 229-239,wherein said glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B,7F, 9V, 14 and 23F are individually conjugated to PD.248. The immunogenic composition of any one of paragraphs 229-239 or247, wherein said glycoconjugate from S. pneumoniae serotype 18C isconjugated to TT.249. The immunogenic composition of any one of paragraphs 229-239 or247-248, wherein said glycoconjugate from S. pneumoniae serotype 19F isconjugated to DT.250. The immunogenic composition of any one of paragraphs 229-239 or247-249, wherein said glycoconjugates from S. pneumoniae serotypes 1, 4,5, 6B, 7F, 9V, 14 and/or 23F are individually conjugated to PD, saidglycoconjugate from S. pneumoniae serotype 18C is conjugated to TT andsaid glycoconjugate from S. pneumoniae serotype 19F is conjugated to DT.251. The immunogenic composition of any one of paragraphs 247-250,wherein said glycoconjugate from S. pneumoniae serotype 22F isconjugated to CRM₁₉₇.252. The immunogenic composition of any one of paragraphs 247-251,wherein said glycoconjugate from S. pneumoniae serotype 33F isconjugated to CRM₁₉₇.253. The immunogenic composition of any one of paragraphs 226-252,wherein said second immunogenic composition is a 7, 8, 9, 10, 11, 12,13, 14 or 15-valent pneumococcal conjugate composition.254. The immunogenic composition of any one of paragraphs 226-253,wherein said second immunogenic composition is a 10, 11, 12, 13, 14 or15-valent pneumococcal conjugate composition.255. The immunogenic composition of any one of paragraphs 226-254,wherein said second immunogenic composition is a 13, 14 or 15-valentpneumococcal conjugate composition.256. The immunogenic composition of any one of paragraphs 226-255,wherein said second immunogenic composition is a 13-valent pneumococcalconjugate composition.257. The immunogenic composition of any one of paragraphs 226-254,wherein said second immunogenic composition is an 11-valent pneumococcalconjugate composition wherein said 11 conjugates consists ofglycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and23F individually conjugated to PD, glycoconjugate from S. pneumoniaeserotype 18C conjugated to TT, glycoconjugate from S. pneumoniaeserotype 19F conjugated to DT and glycoconjugate from S. pneumoniaeserotype 22F conjugated to CRM₁₉₇.258. The immunogenic composition of any one of paragraphs 226-254,wherein said second immunogenic composition is an 11-valent pneumococcalconjugate composition wherein said 11 conjugates consists ofglycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and23F individually conjugated to PD, glycoconjugate from S. pneumoniaeserotype 18C conjugated to TT, glycoconjugate from S. pneumoniaeserotype 19F conjugated to DT and glycoconjugate from S. pneumoniaeserotype 33F conjugated to CRM₁₉₇.259. The immunogenic composition of any one of paragraphs 226-254,wherein said second immunogenic composition is a 12-valent pneumococcalconjugate composition wherein said 12 conjugates consists ofglycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and23F individually conjugated to PD, glycoconjugate from S. pneumoniaeserotype 18C conjugated to TT, glycoconjugate from S. pneumoniaeserotype 19F conjugated to DT, glycoconjugate from S. pneumoniaeserotype 22F conjugated to CRM₁₉₇ and glycoconjugate from S. pneumoniaeserotype 33F conjugated to CRM₁₉₇.260. The immunogenic composition of any one of paragraphs 226-256,wherein said second immunogenic composition is a 13-valent pneumococcalconjugate composition wherein said 13 conjugates consists ofglycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V,14, 18C, 19A, 19F and 23F individually conjugated to CRM₁₉₇.261. The immunogenic composition of any one of paragraphs 226-255,wherein said second immunogenic composition is a 14-valent pneumococcalconjugate composition wherein said 14 conjugates consists ofglycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V,14, 18C, 19A, 19F, 23F and 22F individually conjugated to CRM₁₉₇.262. The immunogenic composition of any one of paragraphs 226-255,wherein said second immunogenic composition is a 14-valent pneumococcalconjugate composition wherein said 14 conjugates consists ofglycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V,14, 18C, 19A, 19F, 23F and 33F individually conjugated to CRM₁₉₇.263. The immunogenic composition of any one of paragraphs 226-255,wherein said second immunogenic composition is a 15-valent pneumococcalconjugate composition wherein said 15 conjugates consists ofglycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V,14, 18C, 19A, 19F, 23F, 22F and 33F individually conjugated to CRM₁₉₇.264. The immunogenic composition of any one of paragraphs 229-263,wherein said glycoconjugates of the second immunogenic composition areall conjugated to the carrier protein by reductive amination.265. The immunogenic composition of any one of paragraphs 229-264,wherein each dose of said second immunogenic composition comprises 1 to10 μg of polysaccharide of each serotype.266. The immunogenic composition of any one of paragraphs 229-265,wherein each dose of said second immunogenic composition comprises 10 μgto 150 μg of carrier protein.267. The immunogenic composition of any one of paragraphs 229-266,wherein each dose of said second immunogenic composition comprises about15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg,about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg,about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg,about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about48 μg, about 49 μg or about 50 μg of carrier protein.268. The immunogenic composition of any one of paragraphs 229-267,wherein said second immunogenic composition further comprise antigensfrom other pathogens.269. The immunogenic composition of any one of paragraphs 229-268,wherein said second immunogenic composition further comprises at leastone adjuvant.270. The immunogenic composition of any one of paragraphs 229-268,wherein said second immunogenic composition further comprises at leastone adjuvant selected from the group consisting of aluminum phosphate,aluminum sulfate and aluminum hydroxide.271. The immunogenic composition of any one of paragraphs 229-268,wherein said second immunogenic composition further comprises aluminumphosphate as adjuvant.272. The immunogenic composition of any one of paragraphs 229-268,wherein said second immunogenic composition further comprises from 0.2mg/mL to 0.3 mg/mL of elemental aluminum in the form of aluminumphosphate as adjuvant.273. The immunogenic composition of any one of paragraphs 229-268,wherein said second immunogenic composition further comprises about 0.25mg/mL of elemental aluminum in the form of aluminum phosphate asadjuvant.274. The immunogenic composition of any one of paragraphs 229-273,wherein said second immunogenic composition further comprises a buffer.275. The immunogenic composition of any one of paragraphs 229-274,wherein said second immunogenic composition comprises a buffer having apKa of about 3.5 to about 7.5.276. The immunogenic composition of any one of paragraphs 274-275,wherein said buffer of said second immunogenic composition is phosphate,succinate, histidine or citrate.277. The immunogenic composition of any one of paragraphs 274-276,wherein said buffer of said second immunogenic composition is succinateat a final concentration of about 5.0 mM.278. The immunogenic composition of any one of paragraphs 229-277,wherein said second immunogenic composition further comprises a salt.279. The immunogenic composition of any one of paragraphs 229-278wherein said salt of said second immunogenic composition is selectedfrom the group consisting of magnesium chloride, potassium chloride,sodium chloride and a combination thereof.280. The immunogenic composition of any one of paragraphs 229-179,wherein said second immunogenic composition comprises sodium chloride ata final concentration of 150 mM.281. The immunogenic composition of any one of paragraphs 229-280,wherein said second immunogenic composition further comprises asurfactant.282. The immunogenic composition of paragraph 281, wherein saidsurfactant of said second immunogenic composition is polysorbate 80.283. The immunogenic composition of any one of paragraphs 281-282,wherein the final concentration of polysorbate 80 in said secondimmunogenic composition is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%,0.07%, 0.08%, 0.09% or 0.1% (w/w).284. The immunogenic composition of any one of paragraphs 229-283,wherein said second immunogenic composition has a pH of 5.8 to 6.0.285. The immunogenic composition of any one of paragraphs 1-165 for usein vaccination wherein the vaccination schedule is a single doseschedule.286. The immunogenic composition of any one of paragraphs 1-165 for usein vaccination wherein the vaccination schedule is a multiple doseschedule.287. The immunogenic composition of any one of paragraphs 1-165 for usein vaccination wherein the vaccination schedule consists of a series of2 doses separated by an interval of about 1 month to about 12 months.288. The immunogenic composition of any one of paragraphs 1-165 for usein vaccination wherein the vaccination schedule consists of a series of2 doses separated by an interval of about 1 month to about 6 months.289. The immunogenic composition of any one of paragraphs 1-165 for usein vaccination wherein the vaccination schedule consists of a series of2 doses separated by an interval of about 1 month to about 2 months290. The immunogenic composition of any one of paragraphs 1-165 for usein vaccination wherein the vaccination schedule consists of a series of3 doses separated by an interval of about 1 month to about 12 months.291. The immunogenic composition of any one of paragraphs 1-165 for usein vaccination wherein the vaccination schedule consists of a series of3 doses separated by an interval of about 1 month to about 6 months.292. The immunogenic composition of any one of paragraphs 1-165 for usein vaccination wherein the vaccination schedule consists of a series of3 doses separated by an interval of about 1 month to about 2 months.293. The immunogenic composition of any one of paragraphs 1-165 for usein vaccination wherein the vaccination schedule consists of a series of3 doses separated by an interval of about 1 month to about 4 monthsfollowed by a fourth dose about 10 months to about 13 months after thefirst dose.294. The immunogenic composition of any one of paragraphs 1-165 for usein vaccination wherein the vaccination schedule consists of a series of3 doses separated by an interval of about 1 month to about 2 monthsfollowed by a fourth dose about 10 months to about 13 months after thefirst dose.295. The immunogenic composition of any one of paragraphs 1-165 for usein vaccination wherein the vaccination schedule consists of a series of2 or 3 doses separated by an interval of about 1 month to about 2months, starting at 2 months of age, followed by a toddler dose at 12-18months of age.296. The immunogenic composition of any one of paragraphs 1-165 for usein vaccination wherein the vaccination schedule consists of a series of2 doses separated by an interval of about 2 months, starting at 2 monthsof age, followed by a toddler dose at 12-18 months of age.297. The immunogenic composition of any one of paragraphs 1-165 for usein vaccination wherein the vaccination schedule consists of a 4 doses ofvaccine administered at 2, 4, 6, and 12-15 months of age.298. The immunogenic composition of any one of paragraphs 1-165 for usein vaccination wherein the vaccination schedule consists of a prime dosegiven at day 0 and one or more booster doses given at intervals thatrange from about 2 to about 24 weeks.299. The kit of any one of paragraphs 166-225 for simultaneous,concurrent, concomitant or sequential administration of the first andsecond immunogenic compositions.300. The immunogenic composition of any one of paragraphs 226-284 or thekit of paragraph 299 for use in a method of simultaneous administrationof the first and second immunogenic compositions.301. The immunogenic composition or kit of paragraph 300 wherein theschedule of vaccination of said simultaneous administration is a singledose.302. The immunogenic composition or kit of paragraph 300 wherein theschedule of vaccination of said simultaneous administration is amultiple dose schedule.303. The immunogenic composition or kit of paragraph 302 wherein saidmultiple dose schedule consists of a series of 2 doses separated by aninterval of about 1 month to about 12 months.304. The immunogenic composition or kit of paragraph 302 wherein saidmultiple dose schedule consists of a series of 2 doses separated by aninterval of about 1 month to about 2 months.305. The immunogenic composition or kit of paragraph 302 wherein saidmultiple dose schedule consists of a series of 3 doses separated by aninterval of about 1 month to about 12 months.306. The immunogenic composition or kit of paragraph 302 wherein saidmultiple dose schedule consists of a series of 3 doses separated by aninterval of about 1 month to about 2 months.307. The immunogenic composition or kit of paragraph 302 wherein saidmultiple dose schedule consists of a series of 3 doses separated by aninterval of about 1 month to about 2 months followed by a fourth doseabout 10 months to about 13 months after the first dose.308. The immunogenic composition or kit of paragraph 302 wherein saidmultiple dose schedule consists of a series of 3 doses wherein each doseis separated by an interval of about 1, 2, 3 or 4 months followed by afourth dose about 10 months to about 13 months after the first dose.309. The immunogenic composition or kit of paragraph 302 wherein saidmultiple dose schedule consists of at least one dose (e.g., 1, 2 or 3doses) in the first year of age followed by at least one toddler dose.310. The immunogenic composition or kit of paragraph 302 wherein saidmultiple dose schedule consists of a series of 2 or 3 doses separated byan interval of about 1 month to about 2 months (for example 28-56 daysbetween doses), starting at 2 months of age, followed by a toddler doseat 12-18 months of age.311. The immunogenic composition or kit of paragraph 302 wherein saidmultiple dose schedule consists of a 4 dose series of vaccineadministered at 2, 4, 6, and 12-15 months of age.312. The immunogenic composition or kit of paragraph 302 wherein saidmultiple dose schedule consists of a prime dose given at day 0 and oneor more booster doses given at intervals that range from about 2 toabout 24 weeks, preferably with a dosing interval of 4-8 weeks.313. The immunogenic composition or kit of paragraph 302 wherein saidmultiple dose schedule consists of a prime dose given at day 0 and abooster dose given about 3 months later.314. The immunogenic composition of any one of paragraphs 226-284 or thekit of paragraph 299 for use in a method of concomitant administrationof the first and second immunogenic compositions.315. The immunogenic composition or kit of paragraph 314 wherein theschedule of vaccination of said concomitant administration is a singledose.316. The immunogenic composition or kit of paragraph 314 wherein theschedule of vaccination of said concomitant administration is a multipledose schedule.317. The immunogenic composition of any one of paragraphs 226-284 or thekit of paragraph 299 for use in a method of concurrent administration ofthe first and second immunogenic compositions.318. The immunogenic composition or kit of paragraph 317 wherein theschedule of vaccination of said concurrent administration is a singledose.319. The immunogenic composition or kit of paragraph 317 wherein theschedule of vaccination of said concurrent administration is a multipledose schedule.320. The immunogenic composition or kit of paragraph 316 or 319 whereinsaid multiple dose schedule consists of a series of 2 doses separated byan interval of about 1 month to about 12 months.321. The immunogenic composition or kit of paragraph 316 or 319 whereinsaid multiple dose schedule consists of a series of 2 doses separated byan interval of about 1 month to about 2 months.322. The immunogenic composition or kit of paragraph 315 or 318 whereinsaid multiple dose schedule consists of a series of 3 doses separated byan interval of about 1 month to about 12 months.323. The immunogenic composition or kit of paragraph 316 or 319 whereinsaid multiple dose schedule consists of a series of 3 doses separated byan interval of about 1 month to about 2 months.324. The immunogenic composition or kit of paragraph 316 or 319 whereinsaid multiple dose schedule consists of a series of 3 doses separated byan interval of about 1 month to about 4 months followed by a fourth doseabout 10 months to about 13 months after the first dose.325. The immunogenic composition or kit of paragraph 316 or 319 whereinsaid multiple dose schedule consists of a series of 3 doses separated byan interval of about 1 month to about 2 months followed by a fourth doseabout 10 months to about 13 months after the first dose.326. The immunogenic composition or kit of paragraph 316 or 319 whereinsaid multiple dose schedule consists of at least one dose (e.g., 1, 2 or3 doses) in the first year of age followed by at least one toddler dose.327. The immunogenic composition or kit of paragraph 316 or 319 whereinsaid multiple dose schedule consists of a series of 2 or 3 dosesseparated by an interval of about 1 month to about 2 months (for example28-56 days between doses), starting at 2 months of age, followed by atoddler dose at 12-18 months of age.328. The immunogenic composition or kit of paragraph 316 or 319 whereinsaid multiple dose schedule consists of a 4-dose series of vaccineadministered at 2, 4, 6, and 12-15 months of age.329. The immunogenic composition or kit of paragraph 316 or 319 whereinsaid multiple dose schedule consists of a prime dose given at day 0 andone or more booster doses given at intervals that range from about 2 toabout 24 weeks, preferably with a dosing interval of 4-8 weeks.330. The immunogenic composition or kit of paragraph 316 or 319 whereinsaid multiple dose schedule consists of a prime dose given at day 0 anda booster dose given about 3 months later.331. The immunogenic composition of any one of paragraphs 226-284 or thekit of paragraph 299 for use in a method of sequential administration ofthe first and second immunogenic compositions.332. The immunogenic composition or kit of paragraph 331 wherein theschedule of vaccination of said sequential administration consists of aseries of 2, 3, 4, 5, 6, 7 or 8 doses.333. The immunogenic composition or kit of paragraph 331 or 332 whereinthe schedule of vaccination of said sequential administration consistsof a series of 2, 3 or 4 doses.334. The immunogenic composition or kit of any one of paragraphs 331-333wherein the first immunogenic composition is administered first and thesecond immunogenic composition is administered second.335. The immunogenic composition or kit of any one of paragraphs 331-333wherein the second immunogenic composition is administered first and thefirst immunogenic composition is administered second.336. The immunogenic composition or kit of any one of paragraphs 331-335wherein the schedule of vaccination consists of a series of 2 dosesseparated by an interval of about 1 month to about 12 months.337. The immunogenic composition or kit of any one of paragraphs 331-335wherein the schedule of vaccination consists of a series of 2 dosesseparated by an interval of about 1 month to about 2 months.338. The immunogenic composition or kit of any one of paragraphs 331-338wherein the first and second doses are administered in the first year ofage.339. The immunogenic composition or kit of any one of paragraphs 331-338wherein the first dose is administered in the first year of age and thesecond dose is a toddler dose.340. The immunogenic composition or kit of paragraph 339 wherein saidtoddler dose is administered at 12-18 months of age.341. The immunogenic composition or kit of paragraph 332 or 333 whereinthe schedule of vaccination of said sequential administration consistsof a series of 3 doses.342. The immunogenic composition or kit of paragraph 341 wherein saidschedule consists of a series of 3 doses wherein each dose is separatedby an interval of about 1 month to about 12 months.343. The immunogenic composition or kit of paragraph 341 wherein saidschedule consists of a series of 3 doses wherein each dose is separatedby an interval of about 1 month to about 2 months.344. The immunogenic composition or kit of any one of paragraphs 341-343wherein the first and second doses are administered in the first year ofage and the third dose is a toddler dose.345. The immunogenic composition or kit of any one of paragraphs 341-344wherein the first and second doses are separated by an interval of about1 month to about 2 months (for example 28-56 days between doses),starting at 2 months of age, and the third dose is a toddler dose at12-18 months of age.346. The immunogenic composition or kit of any one of paragraphs 341-345wherein the first immunogenic composition is administered as the firstand second doses and the second immunogenic composition is administeredas the third dose.347. The immunogenic composition or kit of any one of paragraphs 341-345wherein the second immunogenic composition is administered as the firstand second doses and the first immunogenic composition is administeredas the third dose.348. The immunogenic composition or kit of any one of paragraphs 341-345wherein the first immunogenic composition is administered as the firstdose, the second immunogenic composition is administered as the seconddose and the first immunogenic composition is administered as the thirddose.349. The immunogenic composition or kit of any one of paragraphs 341-345wherein the second immunogenic composition is administered as the firstdose, the first immunogenic composition is administered as the seconddose and the second immunogenic composition is administered as the thirddose.350. The immunogenic composition or kit of any one of paragraphs 341-345wherein the first immunogenic composition is administered as the firstdose and the second immunogenic composition is administered as thesecond and third doses.351. The immunogenic composition or kit of any one of paragraphs 341-345wherein the second immunogenic composition is administered as the firstdose and the first immunogenic composition is administered as the secondand third doses.352. The immunogenic composition or kit of paragraph 332 or 333 whereinthe schedule of vaccination of said sequential administration consistsof a series of 4 doses.353. The immunogenic composition or kit of paragraph 352 wherein thefirst, second and third doses are separated by an interval of about 1month to about 4 months followed by the fourth dose about 10 months toabout 13 months after the first dose.354. The immunogenic composition or kit of paragraph 352 or 353 whereinthe first, second and third doses are separated by an interval of about1 month to about 2 months followed by the fourth dose about 10 months toabout 13 months after the first dose.355. The immunogenic composition or kit of any one of paragraphs 352-354wherein the first, second and third doses are administered in the firstyear of age and the fourth dose is a toddler dose.356. The immunogenic composition or kit of any one of paragraphs 352-355wherein the first, second and third doses are separated by an intervalof about 1 month to about 2 months (for example 28-56 days betweendoses), starting at 2 months of age, and the fourth dose is a toddlerdose at 12-18 months of age.357. The immunogenic composition or kit of any one of paragraphs 352-356wherein the first immunogenic composition is administered as the first,second and third doses and the second immunogenic composition isadministered as the fourth dose.358. The immunogenic composition or kit of any one of paragraphs 352-356wherein the second immunogenic composition is administered as the first,second and third doses and the first immunogenic composition isadministered as the fourth dose.359. The immunogenic composition or kit of any one of paragraphs 352-356wherein the first immunogenic composition is administered as the firstand second doses and the second immunogenic composition is administeredas the third and fourth doses.360. The immunogenic composition or kit of any one of paragraphs 352-356wherein the second immunogenic composition is administered as the firstand second doses and the first immunogenic composition is administeredas the third and fourth doses.361. The immunogenic composition or kit of any one of paragraphs 352-356wherein the first immunogenic composition is administered as the firstand second doses, the second immunogenic composition is administered asthe third dose and the first immunogenic composition is administered asthe fourth dose.362. The immunogenic composition or kit of any one of paragraphs 352-356wherein the second immunogenic composition is administered as the firstand second doses, the first immunogenic composition is administered asthe third dose and the second immunogenic composition is administered asthe fourth dose.363. The immunogenic composition or kit of any one of paragraphs 352-356wherein the first immunogenic composition is administered as the firstdose and the second immunogenic composition is administered as thesecond, third and fourth doses.364. The immunogenic composition or kit of any one of paragraphs 352-356wherein the second immunogenic composition is administered as the firstdose and the first immunogenic composition is administered as thesecond, third and fourth doses.365. The immunogenic composition or kit of any one of paragraphs 352-356wherein the first immunogenic composition is administered as the firstdose, the second immunogenic composition is administered as the seconddose, the first immunogenic composition is administered as the thirddose and the second immunogenic composition is administered as thefourth dose.366. The immunogenic composition or kit of any one of paragraphs 352-356wherein the second immunogenic composition is administered as the firstdose, the first immunogenic composition is administered as the seconddose, the second immunogenic composition is administered as the thirddose and the first immunogenic composition is administered as the fourthdose.367. The immunogenic composition or kit of any one of paragraphs 352-356wherein the first immunogenic composition is administered as the firstdose, the second immunogenic composition is administered as the seconddose and the first immunogenic composition is administered as the thirdand fourth doses.368. The immunogenic composition or kit of any one of paragraphs 352-356wherein the second immunogenic composition is administered as the firstdose, the first immunogenic composition is administered as the seconddose and the second immunogenic composition is administered as the thirdand fourth doses.369. The immunogenic composition or kit of any one of paragraphs 352-356wherein the first immunogenic composition is administered as the firstdose, the second immunogenic composition is administered as the secondand third doses and the first immunogenic composition is administered asthe fourth dose.370. The immunogenic composition or kit of any one of paragraphs 352-356wherein the second immunogenic composition is administered as the firstdose, the first immunogenic composition is administered as the secondand third doses and the second immunogenic composition is administeredas the fourth dose.371. The immunogenic composition or kit of any one of paragraphs 331-332wherein the schedule of vaccination of said sequential administrationconsists of a series of 5 doses.372. The immunogenic composition or kit of paragraph 371 wherein theschedule of vaccination consists of a series of 4 doses separated by aninterval of about 1 month to about 3 months followed by a fifth doseabout 10 months to about 13 months after the first dose.373. The immunogenic composition or kit of any one of paragraphs 371-372wherein the first, second, third and fourth doses are administered inthe first year of age and the fifth dose is a toddler dose.374. The immunogenic composition or kit of any one of paragraphs 371-372wherein the first immunogenic composition (1^(st) IC) and the secondimmunogenic composition (2^(nd) IC) are administered according to any ofthe following schedules:

TABLE E Dose 1 2 3 4 5 2^(nd) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC 1^(st) IC2^(nd) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 2^(nd) IC2^(nd) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd) IC 2^(nd) IC2^(nd) IC 1^(st) IC 1^(st) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC2^(nd) IC 1^(st) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC2^(nd) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 1^(st) IC2^(nd) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 1^(st) IC1^(st) IC 2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC1^(st) IC 1^(st) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 1^(st) IC 1^(st) IC1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 1^(st) IC 1^(st) IC 1^(st) IC1^(st) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 2^(nd) IC2^(nd) IC 2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC2^(nd) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 1^(st) IC 1^(st) IC2^(nd) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 1^(st) IC2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd) IC1^(st) IC 2^(nd) IC 1^(st) IC 1^(st) IC 1^(st) IC 1^(st) IC 1^(st) IC2^(nd) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd) IC 2^(nd) IC1^(st) IC 1^(st) IC 1^(st) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 1^(st) IC1^(st) IC 2^(nd) IC 1^(st) IC 1^(st) IC 1^(st) IC 1^(st) IC 1^(st) IC2^(nd) IC 2^(nd) IC 1^(st) IC 1^(st) IC 1^(st) IC 2^(nd) IC 1^(st) IC1^(st) IC 1^(st) IC 1^(st) IC 1^(st) IC 2^(nd) IC375. The immunogenic composition or kit of any one of paragraphs 331-332wherein the schedule of vaccination of said sequential administrationconsists of a series of 6 doses.376. The immunogenic composition or kit of paragraph 375 wherein theschedule of vaccination consists of a series of 5 doses separated by aninterval of about 1 month to about 2 months followed by a sixth doseabout 10 months to about 13 months after the first dose.377. The immunogenic composition or kit of any one of paragraphs 375-376wherein the first, second, third, fourth and fifth doses areadministered in the first year of age and the sixth dose is a toddlerdose.378. The immunogenic composition or kit of any one of paragraphs 375-377wherein the first immunogenic composition and the second immunogeniccomposition are administered according to any of the schedules ofparagraph 374 followed by a sixth dose.379. The immunogenic composition or kit of any one of paragraphs 378wherein the first immunogenic composition according to the invention isadministered as the sixth dose.380. The immunogenic composition or kit of any one of paragraphs 378wherein the second immunogenic composition according to the invention isadministered as the sixth dose.381. The immunogenic composition or kit of any one of paragraphs 331-332wherein the schedule of vaccination of said sequential administrationconsists of a series of 7 doses.382. The immunogenic composition or kit of paragraph 381 wherein theschedule of vaccination consists of a series of 6 doses separated by aninterval of about 1 month followed by a seventh dose about 10 months toabout 13 months after the first dose.383. The immunogenic composition or kit of any one of paragraphs 381-382wherein the first, second, third, fourth, fifth and sixth doses areadministered in the first year of age and the seventh dose is a toddlerdose.384. The immunogenic composition or kit of any one of paragraphs 381-383wherein the first immunogenic composition and the second immunogeniccomposition are administered according to any of the schedules ofparagraph 379 or 380 followed by a seventh dose.385. The immunogenic composition or kit of paragraph 384 wherein thefirst immunogenic composition according to the invention is administeredas the seventh dose.386. The immunogenic composition or kit of paragraph 384 wherein thesecond immunogenic composition according to the invention isadministered as the seventh dose.387. The immunogenic composition or kit of any one of paragraphs 331-332wherein the schedule of vaccination of said sequential administrationconsists of a series of 8 doses.388. The immunogenic composition or kit of paragraph 387 wherein theschedule of vaccination consists of a series of 7 doses separated by aninterval of about 1 month followed by an eighth dose about 10 months toabout 13 months after the first dose.389. The immunogenic composition or kit of any one of paragraphs 387-388wherein the first, second, third, fourth, fifth, sixth and seventh dosesare administered in the first year of age and the seventh dose is atoddler dose.390. The immunogenic composition or kit of any one of paragraphs 387-389wherein the first immunogenic composition and the second immunogeniccomposition are administered according to any of the schedules ofparagraph 385 or 386 followed by a eighth dose.391. The immunogenic composition or kit of any one of paragraphs 390wherein the first immunogenic composition according to the invention isadministered as the eighth dose.392. The immunogenic composition or kit of any one of paragraphs 390wherein the second immunogenic composition according to the invention isadministered as the eighth dose.393. The immunogenic composition or kit of any one of paragraphs 331-333wherein the schedule of vaccination consists of the sequentialadministration of:(a) the first immunogenic composition and(b) the concomitant or concurrent administration of the firstimmunogenic composition with the second immunogenic composition.394. The immunogenic composition or kit of paragraph 393 wherein theschedule of vaccination consists of a series of 2 administrations.395. The immunogenic composition or kit of any one of paragraphs 393-395wherein the schedule of vaccination consists of a series of 2administrations separated by an interval of about 1 month to about 12months.396. The immunogenic composition or kit of any one of paragraphs 393-396wherein the first immunogenic composition is administered first and theconcomitant or concurrent administration is administered second.397. The immunogenic composition or kit of any one of paragraphs 393-396wherein the concomitant or concurrent administration is administeredfirst and the first immunogenic composition is administered second.398. The immunogenic composition or kit of any one of paragraphs 393-398wherein the first and second administrations are administered in thefirst year of age.399. The immunogenic composition or kit of any one of paragraphs 393-398wherein the first administration is administered in the first year ofage and the second administration is a toddler administration.400. The immunogenic composition or kit of paragraph 399 wherein saidtoddler administration is administered at 12-18 months of age.401. The immunogenic composition or kit of paragraph 393 wherein theschedule of vaccination consists of a series of 3 administrations.402. The immunogenic composition or kit of paragraph 401 wherein saidschedule consists of a series of 3 administrations separated by aninterval of about 1 month to about 12 months.403. The immunogenic composition or kit of any one of paragraphs 401-402wherein the first and second administrations are administered in thefirst year of age and the third administration is a toddleradministration.404. The immunogenic composition or kit of any one of paragraphs 401-403wherein the first and second administrations are separated by aninterval of about 1 month to about 2 months (for example 28-56 daysbetween administrations), starting at 2 months of age, and the thirdadministration is a toddler administration at 12-18 months of age.405. The immunogenic composition or kit of any one of paragraphs 401-404wherein the first immunogenic composition is administered at the firstand second administrations and the concomitant or concurrentadministration is administered at the third administration.406. The immunogenic composition or kit of any one of paragraphs 401-404wherein the concomitant or concurrent administration is administered atthe first and second administrations and the first immunogeniccomposition is administered at the third administration.407. The immunogenic composition or kit of any one of paragraphs 401-404wherein the first immunogenic composition is administered at the firstadministration, the concomitant or concurrent administration isadministered at the second administration and the first immunogeniccomposition is administered at the third administration.408. The immunogenic composition or kit of any one of paragraphs 401-404wherein the concomitant or concurrent administration is administered atthe first administration, the first immunogenic composition isadministered at the second administration and the concomitant orconcurrent is administered at the third administration.409. The immunogenic composition or kit of any one of paragraphs 401-404wherein the first immunogenic composition is administered at the firstadministration and the concomitant or concurrent administration isadministered at the second and third administrations.410. The immunogenic composition or kit of any one of paragraphs 401-404wherein the concomitant or concurrent administration is administered atthe first administration and the first immunogenic composition isadministered at the second and third administrations.411. The immunogenic composition or kit of paragraph 393 wherein theschedule of vaccination consists of a series of 4 administrations.412. The immunogenic composition or kit of paragraph 411 wherein thefirst, second and third administrations are separated by an interval ofabout 1 month to about 4 months followed by the fourth administrationabout 10 months to about 13 months after the first administration.413. The immunogenic composition or kit of paragraph 411 wherein thefirst, second and third administrations are separated by an interval ofabout 1 month to about 2 months followed by the fourth administrationabout 10 months to about 13 months after the first administration.414. The immunogenic composition or kit of any one of paragraphs 411-413wherein the first, second and third administrations are administered inthe first year of age and the fourth administration is a toddleradministration.415. The immunogenic composition or kit of any one of paragraphs 411-414wherein the first, second and third administrations are separated by aninterval of about 1 month to about 2 months (for example 28-56 daysbetween administrations), starting at 2 months of age, and the fourthadministration is a toddler administration at 12-18 months of age.416. The immunogenic composition or kit of any one of paragraphs 411-415wherein the first immunogenic composition is administered at the first,second and third administrations and the concomitant or concurrentadministration is administered at the fourth administration.417. The immunogenic composition or kit of any one of paragraphs 411-415wherein, the concomitant or concurrent administration is administered atthe first, second, and third administrations and the first immunogeniccomposition is administered at the fourth administration.418. The immunogenic composition or kit of any one of paragraphs 411-415wherein the first immunogenic composition is administered at the firstand second administrations and the concomitant or concurrentadministration is administered at the third and fourth administrations.419. The immunogenic composition or kit of any one of paragraphs 411-415wherein the concomitant or concurrent administration is administered atthe first and second administrations and the first immunogeniccomposition is administered at the third and fourth administrations.420. The immunogenic composition or kit of any one of paragraphs 411-415wherein the first immunogenic composition is administered at the firstand second administrations, the concomitant or concurrent administrationis administered at the third administration and the first immunogeniccomposition is administered at the fourth administration.421. The immunogenic composition or kit of any one of paragraphs 411-415wherein the concomitant or concurrent administration is administered atthe first and second administrations, the first immunogenic compositionis administered at the third administration and the concomitant orconcurrent administration is administered at the fourth administration.422. The immunogenic composition or kit of any one of paragraphs 411-415wherein, the first immunogenic composition is administered at the firstadministration and the concomitant or concurrent administration isadministered at the second, third and fourth administrations.423. The immunogenic composition or kit of any one of paragraphs 411-415wherein the concomitant or concurrent administration is administered atthe first administration and the first immunogenic composition isadministered at the second, third and fourth administrations.424. The immunogenic composition or kit of any one of paragraphs 411-415wherein the first immunogenic composition is administered at the firstadministration, the concomitant or concurrent administration isadministered at the second administration, the first immunogeniccomposition is administered at the third administration and theconcomitant or concurrent administration is administered at the fourthadministration.425. The immunogenic composition or kit of any one of paragraphs 411-415wherein the concomitant or concurrent administration is administered atthe first administration, the first immunogenic composition isadministered at the second administration, the concomitant or concurrentadministration is administered at the third administration and the firstimmunogenic composition is administered at the fourth administration.426. The immunogenic composition or kit of any one of paragraphs 411-415wherein the first immunogenic composition is administered at the firstadministration, the concomitant or concurrent administration isadministered at the second administration and the first immunogeniccomposition is administered at the third and fourth administrations.427. The immunogenic composition or kit of any one of paragraphs 411-415wherein the concomitant or concurrent administration is administered atthe first administration, the first immunogenic composition isadministered at the second administration and the concomitant orconcurrent administration is administered at the third and fourthadministrations.428. The immunogenic composition or kit of any one of paragraphs 411-415wherein the first immunogenic composition is administered at the firstadministration, the concomitant or concurrent administration isadministered at the second and third administrations and the firstimmunogenic composition is administered at the fourth administration.429. The immunogenic composition or kit of any one of paragraphs 411-415wherein the concomitant or concurrent administration is administered atthe first administration, the first immunogenic composition isadministered at the second and third administrations and the concomitantor concurrent administration is administered at the fourthadministration.430. The immunogenic composition or kit of paragraph 393 wherein theschedule of vaccination consists of a series of 5 administrations.431. The immunogenic composition or kit of paragraph 430 wherein theschedule consists of a series of 4 administrations wherein each dose isseparated by an interval of about 1 month to about 3 months followed bya fifth administration about 10 months to about 13 months after thefirst administration.432. The immunogenic composition or kit of any one of paragraphs 430-431wherein, the first, second, third and fourth administrations areadministered in the first year of age and the fifth administration is atoddler dose.433. The immunogenic composition or kit of any one of paragraphs 430-432wherein, the first immunogenic composition (1^(st) IC) and theconcomitant or concurrent administration of the first immunogeniccomposition with the second immunogenic composition (1^(st) IC/2^(nd)IC) are administered according to any of the following schedules:

TABLE F Dose 1 2 3 4 5 1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC 1stIC/2nd IC 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC 1^(st) IC1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC 1^(st) IC 1^(st)IC 1st IC/2nd IC 1st IC/2nd IC 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 1stIC/2nd IC 1st IC/2nd IC 1^(st) IC 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC1st IC/2nd IC 1^(st) IC 1^(st) IC 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC1^(st) IC 1st IC/2nd IC 1^(st) IC 1st IC/2nd IC 1^(st) IC 1st IC/2nd IC1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC 1^(st) IC 1st IC/2nd IC 1stIC/2nd IC 1^(st) IC 1st IC/2nd IC 1^(st) IC 1st IC/2nd IC 1^(st) IC 1stIC/2nd IC 1st IC/2nd IC 1^(st) IC 1st IC/2nd IC 1^(st) IC 1^(st) IC 1stIC/2nd IC 1^(st) IC 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC1^(st) IC 1^(st) IC 1st IC/2nd IC 1^(st) IC 1st IC/2nd IC 1^(st) IC1^(st) IC 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 1^(st) IC 1^(st) IC1^(st) IC 1^(st) IC 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC1st IC/2nd IC 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC 1^(st)IC 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 1^(st) IC 1st IC/2nd IC 1^(st)IC 1st IC/2nd IC 1st IC/2nd IC 1^(st) IC 1^(st) IC 1^(st) IC 1st IC/2ndIC 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 1^(st) IC 1st IC/2nd IC 1^(st)IC 1st IC/2nd IC 1^(st) IC 1^(st) IC 1st IC/2nd IC 1^(st) IC 1^(st) IC1st IC/2nd IC 1^(st) IC 1st IC/2nd IC 1^(st) IC 1^(st) IC 1^(st) IC1^(st) IC 1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC 1^(st) IC1^(st) IC 1st IC/2nd IC 1st IC/2nd IC 1^(st) IC 1^(st) IC 1^(st) IC 1stIC/2nd IC 1^(st) IC 1st IC/2nd IC 1^(st) IC 1^(st) IC 1st IC/2nd IC1^(st) IC 1^(st) IC 1^(st) IC 1^(st) IC 1^(st) IC 1st IC/2nd IC 1stIC/2nd IC 1^(st) IC 1^(st) IC 1^(st) IC 1st IC/2nd IC 1^(st) IC 1^(st)IC 1^(st) IC 1^(st) IC 1^(st) IC 1st IC/2nd IC434. The immunogenic composition or kit of paragraph 393 wherein theschedule of vaccination consists of a series of 6 administrations.435. The immunogenic composition or kit of paragraph 434 wherein theschedule consists of a series of 5 administrations wherein eachadministration is separated by an interval of about 1 month to about 2months followed by a sixth administration about 10 months to about 13months after the first administration.436. The immunogenic composition or kit any one of paragraphs 434-435wherein the first, second, third, fourth and fifth administrations areadministered in the first year of age and the sixth administration is atoddler administration.437. The immunogenic composition or kit any one of paragraphs 434-436wherein the first immunogenic composition and the concomitant orconcurrent administration of the first immunogenic composition with thesecond immunogenic composition are administered according to any of theschedules of paragraph 433 followed by a sixth administration.438. The immunogenic composition or kit of any one of paragraphs 437wherein the first immunogenic composition is administered as the sixthadministration.439. The immunogenic composition or kit of any one of paragraphs 437wherein the concomitant or concurrent administration of the firstimmunogenic composition with the second immunogenic composition isadministered at the sixth administration.440. The immunogenic composition or kit of paragraph 393 wherein theschedule of vaccination consists of a series of 7 administrations.441. The immunogenic composition or kit of paragraph 440 wherein theschedule of vaccination consists of a series of 6 administrationswherein each administration is separated by an interval of about 1 monthfollowed by a seventh administration about 10 months to about 13 monthsafter the first administration.442. The immunogenic composition or kit of any one of paragraphs 440-441wherein, the first, second, third, fourth, fifth and sixthadministrations are administered in the first year of age and theseventh administration is a toddler administration.443. The immunogenic composition or kit of any one of paragraphs 440-442wherein the first immunogenic composition and the concomitantadministration of the first immunogenic composition with the secondimmunogenic composition are administered according to any of theschedule of paragraph 438 or 439 followed by a seventh administration.444. The immunogenic composition or kit of paragraph 443 wherein thefirst immunogenic composition is administered as the seventhadministration.445. The immunogenic composition or kit of paragraph 443 wherein theconcomitant or concurrent administration of the first immunogeniccomposition with the second immunogenic composition is administered asthe seventh administration.446. The immunogenic composition or kit of paragraph 393 wherein theschedule of vaccination consists of a series of 8 administrations.447. The immunogenic composition or kit of paragraph 446 wherein theschedule of vaccination consists of a series of 7 administrationswherein each administration is separated by an interval of about 1 monthfollowed by an eighth administration about 10 months to about 13 monthsafter the first administration.448. The immunogenic composition or kit of any one of paragraphs 446-447wherein, the first, second, third, fourth, fifth, sixth and seventhadministrations are administered in the first year of age and theseventh administration is a toddler administration.449. The immunogenic composition or kit of any one of paragraphs 446-448wherein the first immunogenic composition and the concomitant orconcurrent administration of the first immunogenic composition with thesecond immunogenic composition are administered according to any of theschedule of paragraph 444 or 445 followed by an eighth administration.450. The immunogenic composition or kit of paragraph 449 wherein thefirst immunogenic composition is administered as the eighthadministration.451. The immunogenic composition or kit of paragraph 449 wherein theconcomitant or concurrent administration of the first immunogeniccomposition with the second immunogenic composition is administered asthe eighth administration.452. The immunogenic composition or kit of any one of paragraphs 331-333wherein the schedule of vaccination consists of the sequentialadministration of:(a) the second immunogenic composition and(b) the concomitant or concurrent administration of the firstimmunogenic composition with the second immunogenic composition453. The immunogenic composition or kit of paragraph 452 wherein saidschedule is any one of the schedule according to paragraphs 394-451wherein administration of said second immunogenic composition of (a)replaces administration of the first immunogenic composition of (a) insaid paragraphs.454. The immunogenic composition of any one of paragraphs 1-165 or thekit of any one of paragraphs 166-225 for use as a medicament.455. The immunogenic composition of any one of paragraphs 1-165 or thekit of any one of paragraphs 166-225 for use as a vaccine.456. The immunogenic composition of any one of paragraphs 1-165 or thekit of any one of paragraphs 166-225 for use in a method for preventing,treating or ameliorating a bacterial infection, disease or condition ina subject.457. The immunogenic composition of any one of paragraphs 1-165 or thekit of any one of paragraphs 166-225 for use in a method for preventinga bacterial infection, disease or condition in a subject.458. The immunogenic composition of any one of paragraphs 1-165 or thekit of any one of paragraphs 166-225 for use in a method to protect ortreat a human susceptible to pneumococcal infection, by means ofadministering said immunogenic compositions via a systemic or mucosalroute.459. The immunogenic composition or kit of paragraph 458 wherein saidimmunogenic composition(s) is/are administered by intramuscular,intraperitoneal, intradermal or subcutaneous routes.460. The immunogenic composition of any one of paragraphs 1-165 or thekit of any one of paragraphs 166-225 for use as a vaccine, wherein thesubject to be vaccinated is human being less than 1 year of age.461. The immunogenic composition of any one of paragraphs 1-165 or thekit of any one of paragraphs 166-225 for use as a vaccine, wherein thesubject to be vaccinated is a human being less than 2 year of age.462. The immunogenic composition of any one of paragraphs 1-165 or thekit of any one of paragraphs 166-225 for use as a vaccine, wherein thesubject to be vaccinated is a human adult 50 years of age or older.463. The immunogenic composition of any one of paragraphs 1-165 or thekit of any one of paragraphs 166-225 for use as a vaccine, wherein thesubject to be vaccinated is an immunocompromised human.464. The immunogenic composition of any one of paragraphs 1-165 or thekit of any one of paragraphs 166-225 for use in a single dose schedule.465. The immunogenic composition of any one of paragraphs 1-165 or thekit of any one of paragraphs 166-225 for use in a multiple doseschedule.466. The immunogenic composition or kit of paragraph 465 wherein saidmultiple dose schedule consists of a series of 2 doses separated by aninterval of about 1 month to about 2 months.467. The immunogenic composition or kit of paragraph 465 wherein saidmultiple dose schedule consists of a series of 3 doses separated by aninterval of about 1 month to about 2 months.468. The immunogenic composition or kit of paragraph 465 wherein saidmultiple dose schedule consists of a series of 3 doses separated by aninterval of about 1 month to about 2 months followed by a fourth doseabout 10 months to about 13 months after the first dose.469. The immunogenic composition or kit of paragraph 465 wherein saidmultiple dose schedule consists of at least one dose in the first yearof age followed by at least one toddler dose.470. The immunogenic composition or kit of paragraph 465 wherein saidmultiple dose schedule consists of a series of 2 or 3 doses separated byan interval of about 1 month to about 2 months, starting at 2 months ofage, and followed by a toddler dose at 12-18 months of age.471. The immunogenic composition or kit of paragraph 465 wherein saidmultiple dose schedule consists of 4 doses series of vaccineadministered at 2, 4, 6, and 12-15 months of age.472. The immunogenic composition or kit of any one of paragraphs 331-333wherein the schedule of vaccination consists of the sequentialadministration of:(a) the second immunogenic composition and(b) the concomitant or concurrent administration of the firstimmunogenic composition with the second immunogenic composition.473. The immunogenic composition or kit of paragraph 472 wherein theschedule of vaccination consists of a series of 2 administrations.474. The immunogenic composition or kit of any one of paragraphs 472-473wherein the schedule of vaccination consists of a series of 2administrations separated by an interval of about 1 month to about 12months.475. The immunogenic composition or kit of any one of paragraphs 472-474wherein the second immunogenic composition is administered first and theconcomitant or concurrent administration is administered second.476. The immunogenic composition or kit of any one of paragraphs 472-474wherein the concomitant or concurrent administration is administeredfirst and the second immunogenic composition is administered second.477. The immunogenic composition or kit of any one of paragraphs 472-476wherein the first and second administrations are administered in thefirst year of age.478. The immunogenic composition or kit of any one of paragraphs 472-476wherein the first administration is administered in the first year ofage and the second administration is a toddler administration.479. The immunogenic composition or kit of paragraph 478 wherein saidtoddler administration is administered at 12-18 months of age.480. The immunogenic composition or kit of paragraph 472 wherein theschedule of vaccination consists of a series of 3 administrations.481. The immunogenic composition or kit of paragraph 480 wherein saidschedule consists of a series of 3 administrations separated by aninterval of about 1 month to about 12 months.482. The immunogenic composition or kit of any one of paragraphs 480-481wherein the first and second administrations are administered in thefirst year of age and the third administration is a toddleradministration.483. The immunogenic composition or kit of any one of paragraphs 480-482wherein the first and second administrations are separated by aninterval of about 1 month to about 2 months (for example 28-56 daysbetween administrations), starting at 2 months of age, and the thirdadministration is a toddler administration at 12-18 months of age.484. The immunogenic composition or kit of any one of paragraphs 480-483wherein the second immunogenic composition is administered at the firstand second administrations and the concomitant or concurrentadministration is administered at the third administration.485. The immunogenic composition or kit of any one of paragraphs 480-483wherein the concomitant or concurrent administration is administered atthe first and second administrations and the second immunogeniccomposition is administered at the third administration.486. The immunogenic composition or kit of any one of paragraphs 480-483wherein the second immunogenic composition is administered at the firstadministration, the concomitant or concurrent administration isadministered at the second administration and the second immunogeniccomposition is administered at the third administration.487. The immunogenic composition or kit of any one of paragraphs 480-483wherein the concomitant or concurrent administration is administered atthe first administration, the second immunogenic composition isadministered at the second administration and the concomitant orconcurrent is administered at the third administration.488. The immunogenic composition or kit of any one of paragraphs 480-483wherein the second immunogenic composition is administered at the firstadministration and the concomitant or concurrent administration isadministered at the second and third administrations.489. The immunogenic composition or kit of any one of paragraphs 480-483wherein the concomitant or concurrent administration is administered atthe first administration and the second immunogenic composition isadministered at the second and third administrations.490. The immunogenic composition or kit of paragraph 472 wherein theschedule of vaccination consists of a series of 4 administrations.491. The immunogenic composition or kit of paragraph 490 wherein thefirst, second and third administrations are separated by an interval ofabout 1 month to about 4 months followed by the fourth administrationabout 10 months to about 13 months after the first administration.492. The immunogenic composition or kit of paragraph 490 wherein thefirst, second and third administrations are separated by an interval ofabout 1 month to about 2 months followed by the fourth administrationabout 10 months to about 13 months after the first administration.493. The immunogenic composition or kit of any one of paragraphs 490-492wherein the first, second and third administrations are administered inthe first year of age and the fourth administration is a toddleradministration.494. The immunogenic composition or kit of any one of paragraphs 490-493wherein the first, second and third administrations are separated by aninterval of about 1 month to about 2 months (for example 28-56 daysbetween administrations), starting at 2 months of age, and the fourthadministration is a toddler administration at 12-18 months of age.495. The immunogenic composition or kit of any one of paragraphs 490-494wherein the second immunogenic composition is administered at the first,second and third administrations and the concomitant or concurrentadministration is administered at the fourth administration.496. The immunogenic composition or kit of any one of paragraphs 490-494wherein, the concomitant or concurrent administration is administered atthe first, second, and third administrations and the second immunogeniccomposition is administered at the fourth administration.497. The immunogenic composition or kit of any one of paragraphs 490-494wherein the second immunogenic composition is administered at the firstand second administrations and the concomitant or concurrentadministration is administered at the third and fourth administrations.498. The immunogenic composition or kit of any one of paragraphs 490-494wherein the concomitant or concurrent administration is administered atthe first and second administrations and the second immunogeniccomposition is administered at the third and fourth administrations.499. The immunogenic composition or kit of any one of paragraphs 490-494wherein the second immunogenic composition is administered at the firstand second administrations, the concomitant or concurrent administrationis administered at the third administration and the second immunogeniccomposition is administered at the fourth administration.500. The immunogenic composition or kit of any one of paragraphs 490-494wherein the concomitant or concurrent administration is administered atthe first and second administrations, the second immunogenic compositionis administered at the third administration and the concomitant orconcurrent administration is administered at the fourth administration.501. The immunogenic composition or kit of any one of paragraphs 490-494wherein, the second immunogenic composition is administered at the firstadministration and the concomitant or concurrent administration isadministered at the second, third and fourth administrations.502. The immunogenic composition or kit of any one of paragraphs 490-494wherein the concomitant or concurrent administration is administered atthe first administration and the second immunogenic composition isadministered at the second, third and fourth administrations.503. The immunogenic composition or kit of any one of paragraphs 490-494wherein the second immunogenic composition is administered at the firstadministration, the concomitant or concurrent administration isadministered at the second administration, the second immunogeniccomposition is administered at the third administration and theconcomitant or concurrent administration is administered at the fourthadministration.504. The immunogenic composition or kit of any one of paragraphs 490-494wherein the concomitant or concurrent administration is administered atthe first administration, the second immunogenic composition isadministered at the second administration, the concomitant or concurrentadministration is administered at the third administration and thesecond immunogenic composition is administered at the fourthadministration.505. The immunogenic composition or kit of any one of paragraphs 490-494wherein the second immunogenic composition is administered at the firstadministration, the concomitant or concurrent administration isadministered at the second administration and the second immunogeniccomposition is administered at the third and fourth administrations.506. The immunogenic composition or kit of any one of paragraphs 490-494wherein the concomitant or concurrent administration is administered atthe first administration, the second immunogenic composition isadministered at the second administration and the concomitant orconcurrent administration is administered at the third and fourthadministrations.507. The immunogenic composition or kit of any one of paragraphs 490-494wherein the second immunogenic composition is administered at the firstadministration, the concomitant or concurrent administration isadministered at the second and third administrations and the secondimmunogenic composition is administered at the fourth administration.508. The immunogenic composition or kit of any one of paragraphs 490-494wherein the concomitant or concurrent administration is administered atthe first administration, the second immunogenic composition isadministered at the second and third administrations and the concomitantor concurrent administration is administered at the fourthadministration.509. The immunogenic composition or kit of paragraph 472 wherein theschedule of vaccination consists of a series of 5 administrations.510. The immunogenic composition or kit of paragraph 509 wherein theschedule consists of a series of 4 administrations wherein each dose isseparated by an interval of about 1 month to about 3 months followed bya fifth administration about 10 months to about 13 months after thefirst administration.511. The immunogenic composition or kit of any one of paragraphs 509-510wherein, the first, second, third and fourth administrations areadministered in the first year of age and the fifth administration is atoddler dose.512. The immunogenic composition or kit of any one of paragraphs 509-511wherein, the second immunogenic composition (2^(nd) IC) and theconcomitant or concurrent administration of the first immunogeniccomposition with the second immunogenic composition (1^(st) IC/2^(nd)IC) are administered according to any of the following schedules:

TABLE G Dose 1 2 3 4 5 1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC 1stIC/2nd IC 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC 2^(nd)IC 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 1stIC/2nd IC 1st IC/2nd IC 2^(nd) IC 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC1st IC/2nd IC 2^(nd) IC 2^(nd) IC 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC2^(nd) IC 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 1stIC/2nd IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 1stIC/2nd IC 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 2^(nd) IC 1stIC/2nd IC 2^(nd) IC 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC2^(nd) IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC2^(nd) IC 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC 2^(nd) IC2^(nd) IC 2^(nd) IC 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC 2^(nd)IC 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 2^(nd)IC 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC 2^(nd) IC 2^(nd) IC 1st IC/2ndIC 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 2^(nd)IC 1st IC/2nd IC 2^(nd) IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 2^(nd) IC1st IC/2nd IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 2^(nd) IC 2^(nd) IC2^(nd) IC 2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC2^(nd) IC 1st IC/2nd IC 1st IC/2nd IC 2^(nd) IC 2^(nd) IC 2^(nd) IC 1stIC/2nd IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 2^(nd) IC 1st IC/2nd IC2^(nd) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC 1st IC/2nd IC 1stIC/2nd IC 2^(nd) IC 2^(nd) IC 2^(nd) IC 1st IC/2nd IC 2^(nd) IC 2^(nd)IC 2^(nd) IC 2^(nd) IC 2^(nd) IC 1st IC/2nd IC513. The immunogenic composition or kit of paragraph 472 wherein theschedule of vaccination consists of a series of 6 administrations.514. The immunogenic composition or kit of paragraph 513 wherein theschedule consists of a series of 5 administrations wherein eachadministration is separated by an interval of about 1 month to about 2months followed by a sixth administration about 10 months to about 13months after the first administration.515. The immunogenic composition or kit any one of paragraphs 513-514wherein the first, second, third, fourth and fifth administrations areadministered in the first year of age and the sixth administration is atoddler administration.516. The immunogenic composition or kit any one of paragraphs 513-515wherein the second immunogenic composition and the concomitant orconcurrent administration of the first immunogenic composition with thesecond immunogenic composition are administered according to any of theschedules of paragraph 512 followed by a sixth administration.517. The immunogenic composition or kit of any one of paragraphs 516wherein the second immunogenic composition is administered as the sixthadministration.518. The immunogenic composition or kit of any one of paragraphs 516wherein the concomitant or concurrent administration of the firstimmunogenic composition with the second immunogenic composition isadministered at the sixth administration.519. The immunogenic composition or kit of paragraph 472 wherein theschedule of vaccination consists of a series of 7 administrations.520. The immunogenic composition or kit of paragraph 519 wherein theschedule of vaccination consists of a series of 6 administrationswherein each administration is separated by an interval of about 1 monthfollowed by a seventh administration about 10 months to about 13 monthsafter the first administration.521. The immunogenic composition or kit of any one of paragraphs 519-520wherein, the first, second, third, fourth, fifth and sixthadministrations are administered in the first year of age and theseventh administration is a toddler administration.522. The immunogenic composition or kit of any one of paragraphs 519-521wherein the second immunogenic composition and the concomitantadministration of the first immunogenic composition with the secondimmunogenic composition are administered according to any of theschedule of paragraph 517 or 518 followed by a seventh administration.523. The immunogenic composition or kit of paragraph 522 wherein thesecond immunogenic composition is administered as the seventhadministration.524. The immunogenic composition or kit of paragraph 522 wherein theconcomitant or concurrent administration of the first immunogeniccomposition with the second immunogenic composition is administered asthe seventh administration.525. The immunogenic composition or kit of paragraph 472 wherein theschedule of vaccination consists of a series of 8 administrations.526. The immunogenic composition or kit of paragraph 525 wherein theschedule of vaccination consists of a series of 7 administrationswherein each administration is separated by an interval of about 1 monthfollowed by an eighth administration about 10 months to about 13 monthsafter the first administration.527. The immunogenic composition or kit of any one of paragraphs 525-526wherein, the first, second, third, fourth, fifth, sixth and seventhadministrations are administered in the first year of age and theseventh administration is a toddler administration.528. The immunogenic composition or kit of any one of paragraphs 525-527wherein the second immunogenic composition and the concomitant orconcurrent administration of the first immunogenic composition with thesecond immunogenic composition are administered according to any of theschedule of paragraph 523 or 524 followed by an eighth administration.529. The immunogenic composition or kit of paragraph 528 wherein thesecond immunogenic composition is administered as the eighthadministration.530. The immunogenic composition or kit of paragraph 528 wherein theconcomitant or concurrent administration of the first immunogeniccomposition with the second immunogenic composition is administered asthe eighth administration.531. The immunogenic composition or kit of paragraph 352 wherein thefirst and second doses are administered in the first year of age and thethird and fourth doses are a toddler dose.532. The immunogenic composition or kit of paragraph 352 wherein theschedule consists of a series of 2 doses wherein each dose is separatedby an interval of about 1 month to about 2 months followed by a thirddose about 10 months to about 13 months after the first dose and afourth dose about 1 month to about 2 months after the third dose.533. The immunogenic composition or kit of any one of paragraphs 531-532wherein the first immunogenic composition is administered as the first,second and third doses and the second immunogenic composition isadministered as the fourth dose.534. The immunogenic composition or kit of any one of paragraphs 531-532wherein the second immunogenic composition is administered as the first,second and third doses and the first immunogenic composition isadministered as the fourth dose.535. The immunogenic composition or kit of any one of paragraphs 531-532wherein the first immunogenic composition is administered as the firstand second doses and the second immunogenic composition is administeredas the third and fourth doses.536. The immunogenic composition or kit of any one of paragraphs 531-532wherein the second immunogenic composition is administered as the firstand second doses and the first immunogenic composition is administeredas the third and fourth doses.537. The immunogenic composition or kit of any one of paragraphs 531-532wherein the first immunogenic composition is administered as the firstand second doses, the second immunogenic composition is administered asthe third dose and the first immunogenic composition is administered asthe fourth dose.538. The immunogenic composition or kit of any one of paragraphs 531-532wherein the second immunogenic composition is administered as the firstand second doses, the first immunogenic composition is administered asthe third dose and the second immunogenic composition is administered asthe fourth dose.539. The immunogenic composition or kit of any one of paragraphs 531-532wherein the first immunogenic composition is administered as the firstdose and the second immunogenic composition is administered as thesecond, third and fourth doses.540. The immunogenic composition or kit of any one of paragraphs 531-532wherein the second immunogenic composition is administered as the firstdose and the first immunogenic composition is administered as thesecond, third and fourth doses.541. The immunogenic composition or kit of any one of paragraphs 531-532wherein the first immunogenic composition is administered as the firstdose, the second immunogenic composition is administered as the seconddose, the first immunogenic composition is administered as the thirddose and the second immunogenic composition is administered as thefourth dose.542. The immunogenic composition or kit of any one of paragraphs 531-532wherein the second immunogenic composition is administered as the firstdose, the first immunogenic composition is administered as the seconddose, the second immunogenic composition is administered as the thirddose and the first immunogenic composition is administered as the fourthdose.543. The immunogenic composition or kit of any one of paragraphs 531-532wherein the first immunogenic composition is administered as the firstdose, the second immunogenic composition is administered as the seconddose and the first immunogenic composition is administered as the thirdand fourth doses.544. The immunogenic composition or kit of any one of paragraphs 531-532wherein the second immunogenic composition is administered as the firstdose, the first immunogenic composition is administered as the seconddose and the second immunogenic composition is administered as the thirdand fourth doses.545. The immunogenic composition or kit of any one of paragraphs 531-532wherein the first immunogenic composition is administered as the firstdose, the second immunogenic composition is administered as the secondand third doses and the first immunogenic composition is administered asthe fourth dose.546. The immunogenic composition or kit of any one of paragraphs 531-532wherein the second immunogenic composition is administered as the firstdose, the first immunogenic composition is administered as the secondand third doses and the second immunogenic composition is administeredas the fourth dose.547. The immunogenic composition or kit of paragraph 371 wherein thefirst, second and third doses are administered in the first year of ageand the fourth and fifth doses are a toddler dose.548. The immunogenic composition or kit of paragraph 371 wherein theschedule consists of a series of 3 doses wherein each dose is separatedby an interval of about 1 month to about 2 months followed by a fourthdose about 10 months to about 13 months after the first dose and a fifthdose about 1 month to about 2 months after the fourth dose.549. The immunogenic composition or kit of any one of paragraphs 547-548wherein the first immunogenic composition (1^(st) IC) and the secondimmunogenic composition (2^(nd) IC) are administered according to any ofthe following schedules:

TABLE H Dose 1 2 3 4 5 2^(nd) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC 1^(st) IC2^(nd) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 2^(nd) IC2^(nd) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd) IC 2^(nd) IC2^(nd) IC 1^(st) IC 1^(st) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC2^(nd) IC 1^(st) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC2^(nd) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 1^(st) IC2^(nd) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 1^(st) IC1^(st) IC 2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC1^(st) IC 1^(st) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 1^(st) IC 1^(st) IC1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 1^(st) IC 1^(st) IC 1^(st) IC1^(st) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 2^(nd) IC2^(nd) IC 2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC2^(nd) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 1^(st) IC 1^(st) IC2^(nd) IC 1^(st) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 1^(st) IC2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd) IC1^(st) IC 2^(nd) IC 1^(st) IC 1^(st) IC 1^(st) IC 1^(st) IC 1^(st) IC2^(nd) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 1^(st) IC 2^(nd) IC 2^(nd) IC1^(st) IC 1^(st) IC 1^(st) IC 2^(nd) IC 1^(st) IC 2^(nd) IC 1^(st) IC1^(st) IC 2^(nd) IC 1^(st) IC 1^(st) IC 1^(st) IC 1^(st) IC 1^(st) IC2^(nd) IC 2^(nd) IC 1^(st) IC 1^(st) IC 1^(st) IC 2^(nd) IC 1^(st) IC1^(st) IC 1^(st) IC 1^(st) IC 1^(st) IC 2^(nd) IC550. The immunogenic composition or kit of any one of paragraphs 547-548wherein the first immunogenic composition (1^(st) IC) and the secondimmunogenic composition (2^(nd) IC) are administered according to any ofthe following schedules:

TABLE J Dose 1 2 3 4 5 2^(nd) IC 2^(nd) IC 2^(nd) IC 2^(nd) IC 1^(st) IC2^(nd) IC 2^(nd) IC 2^(nd) IC 1^(st) IC 2^(nd) IC

As used herein, the term “about” means within a statistically meaningfulrange of a value, such as a stated concentration range, time frame,molecular weight, temperature or pH. Such a range can be within an orderof magnitude, typically within 20%, more typically within 10%, and evenmore typically within 5% or within 1% of a given value or range.Sometimes, such a range can be within the experimental error typical ofstandard methods used for the measurement and/or determination of agiven value or range. The allowable variation encompassed by the term“about” will depend upon the particular system under study, and can bereadily appreciated by one of ordinary skill in the art. Whenever arange is recited within this application, every whole number integerwithin the range is also contemplated as an embodiment of thedisclosure.

The terms “comprising”, “comprise” and “comprises” herein are intendedby the inventors to be optionally substitutable with the terms“consisting essentially of”, “consist essentially of”, “consistsessentially of”, “consisting of”, “consist of” and “consists of”,respectively, in every instance.

An “immunogenic amount”, an “immunologically effective amount”, a“therapeutically effective amount”, a “prophylactically effectiveamount”, or “dose”, each of which is used interchangeably herein,generally refers to the amount of antigen or immunogenic compositionsufficient to elicit an immune response, either a cellular (T cell) orhumoral (B cell or antibody) response, or both, as measured by standardassays known to one skilled in the art.

All references or patent applications cited within this patentspecification are incorporated by reference herein.

The invention is illustrated in the accompanying examples. The examplesbelow are carried out using standard techniques, which are well knownand routine to those of skill in the art, except where otherwisedescribed in detail. The examples are illustrative, but do not limit theinvention.

EXAMPLE Example 1. General Process for Preparation of eTEC LinkedGlycoconjugates

Activation of Saccharide and Thiolation with Cystamine Dihydrochloride

The saccharide is reconstituted in anhydrous dimethylsulfoxide (DMSO).Moisture content of the solution is determined by Karl Fischer (KF)analysis and adjusted to reach a moisture content of between 0.1% and0.4%, typically 0.2%.

To initiate the activation, a solution of1,1′-carbonyl-di-1,2,4-triazole (CDT) or 1,1′-carbonyldiimidazole (CDI)is freshly prepared at a concentration of 100 mg/mL in DMSO. Thesaccharide is activated with various amounts of CDT/CDI (1-10 molarequivalents) and the reaction is allowed to proceed for 1 hour at 23±2°C. The activation level may be determined by HPLC. Cystaminedihydrochloride is freshly prepared in anhydrous DMSO at a concentrationof 50 mg/mL. The activated saccharide is reacted with 1 molarequivalents (mol. eq.) of cystamine dihydrochloride.

Alternatively, the activated saccharide is reacted with 1 mol. eq. ofcysteamine hydrochloride. The thiolation reaction is allowed to proceedfor 21±2 hours at 23±2° C., to produce a thiolated saccharide. Thethiolation level is determined by the added amount of CDT/CDI.

Residual CDT/CDI in the activation reaction solution is quenched by theaddition of 100 mM sodium tetraborate, pH 9.0 solution. Calculations areperformed to determine the added amount of tetraborate and to adjust thefinal moisture content to be up to 1-2% of total aqueous.

Reduction and Purification of Activated Thiolated Saccharide

The thiolated saccharide reaction mixture is diluted 10-fold by additionto pre-chilled 5 mM sodium succinate in 0.9% saline, pH 6.0 and filteredthrough a 5 μm filter. Dialfiltration of thiolated saccharide isperformed against 40-fold diavolume of WFI. To the retentate a solutionof tris(2-carboxyethyl)phosphine (TCEP), 1-5 mol. eq., is added afterdilution by 10% volume of 0.1M sodium phosphate buffer, pH 6.0. Thisreduction reaction is allowed to proceed for 20±2 hours at 5±3° C.Purification of the activated thiolated saccharide is performedpreferably by ultrafiltration/dialfiltration of against pre-chilled 10mM sodium phosphate monobasic, pH 4.3. Alternatively, the thiolatedsaccharide is purified by standard size exclusion chromatographic (SEC)procedures or ion exchange chromatographic methods. An aliquot ofactivated thiolated saccharide retentate is pulled to determine thesaccharide concentration and thiol content (Ellman) assays.

Alternative Reduction and Purification of Activated Thiolated Saccharide

As an alternative to the purification procedure described above,activated thiolated saccharide was also purified as below.

To the thiolated saccharide reaction mixture a solution oftris(2-carboxyethyl)phosphine (TCEP), 5-10 mol. eq., was added andallowed to proceed for 3±1 hours at 23±2° C. The reaction mixture wasthen diluted 5-fold by addition to pre-chilled 5 mM sodium succinate in0.9% saline, pH 6.0 and filtered through a 5 μm filter. Dialfiltrationof thiolated saccharide was performed using 40-fold diavolume ofpre-chilled 10 mM sodium phosphate monobasic, pH 4.3. An aliquot ofactivated thiolated saccharide retentate was pulled to determine thesaccharide concentration and thiol content (Ellman) assays.

Activation and Purification of Bromoacetylated Carrier Protein

Free amino groups of the carrier protein are bromoacteylated by reactionwith a bromoacetylating agent, such as bromoacetic acidN-hydroxysuccinimide ester (BAANS), bromoacetylbromide, or anothersuitable reagent.

The carrier protein (in 0.1M Sodium Phosphate, pH 8.0±0.2) is first keptat 8+3° C., at about pH 7 prior to activation. To the protein solution,the N-hydroxysuccinimide ester of bromoacetic acid (BAANS) as a stockdimethylsulfoxide (DMSO) solution (20 mg/mL) is added in a ratio of0.25-0.5 BAANS: protein (w/w). The reaction is gently mixed at 5+3° C.for 30-60 minutes. The resulting bromoacetylated (activated) protein ispurified, e.g., by ultrafiltration/diafiltration using 10 kDa MWCOmembrane using 10 mM phosphate (pH 7.0) buffer. Following purification,the protein concentration of the bromoacetylated carrier protein isestimated by Lowry protein assay.

The extent of activation is determined by total bromide assay byion-exchange liquid chromatography coupled with suppressed conductivitydetection (ion chromatography). The bound bromide on the activatedbromoacetylated protein is cleaved from the protein in the assay samplepreparation and quantitated along with any free bromide that may bepresent. Any remaining covalently bound bromine on the protein isreleased by conversion to ionic bromide by heating the sample inalkaline 2-mercaptoethanol.

Activation and Purification of Bromoacetylated CRM₁₉₇

CRM₁₉₇ was diluted to 5 mg/mL with 10 mM phosphate buffered 0.9% NaCl pH7 (PBS) and then made 0.1M NaHCO₃, pH 7.0, using 1M stock solution.BAANS was added at a CRM₁₉₇:BAANS ratio 1:0.35 (w:w) using a BAANS stocksolution of 20 mg/mL DMSO. The reaction mixture was incubated at between3° C. and 11° C. for 30 mins-1 hour then purified byultrafiltration/diafiltration using a 10K MWCO membrane and 10 mM SodiumPhosphate/0.9% NaCl, pH 7.0. The purified activated CRM₁₉₇ was assayedby the Lowry assay to determine the protein concentration and thendiluted with PBS to 5 mg/mL. Sucrose was added to 5% wt/vol as acryoprotectant and the activated protein was frozen and stored at −25°C. until needed for conjugation.

Bromoacetylation of lysine residues of CRM₁₉₇ was very consistent,resulting in the activation of 15 to 25 lysines from 39 lysinesavailable. The reaction produced high yields of activated protein.

Conjugation of Activated Thiolated Saccharide to Bromoacetylated CarrierProtein

Before starting the conjugation reaction, the reaction vessels arepre-cooled to 5° C. Bromoacetylated carrier protein and activatedthiolated saccharide are subsequently added and mixed at an agitationspeed of 150-200 rpm. The saccharide/protein input ratio is 0.9±0.1. Thereaction pH is adjusted to 8.0±0.1 with 1M NaOH solution. Theconjugation reaction is allowed to proceed at 5° C. for 20±2 hours.

Capping of Residual Reactive Functional Groups

The unreacted bromoacetylated residues on the carrier protein arequenched by reacting with 2 mol. eq. of N-acetyl-L-cysteine as a cappingreagent for 3 hours at 5° C. Residual free sulfhydryl groups are cappedwith 4 mol. eq. of iodoacetamide (IAA) for 20 hours at 5° C.

Purification of eTEC-Linked Glycoconjugate

The conjugation reaction (post-IAA-capped) mixture is filtered through0.45 μm filter. Ultrafiltration/dialfiltration of the glycoconjugate isperformed against 5 mM succinate-0.9% saline, pH 6.0. The glycoconjugateretentate is then filtered through 0.2 μm filter. An aliquot ofglycoconjugate is pulled for assays. The remaining glycoconjugate isstored at 5° C.

Example 2. Preparation of Pn-33F eTEC Conjugates Activation Process

Activation of Pn33F Polysaccharide

Pn-33F polysaccharide was compounded with 500 mM of 1,2,4-triazole (inWFI) to obtain 10 grams of triazole per gram of polysaccharide. Themixture was shell-frozen in dry ice-ethanol bath and then lyophilized todryness. The lyophilized 33F polysaccharide was reconstituted inanhydrous dimethylsulfoxide (DMSO). Moisture content of the lyophilized33F/DMSO solution was determined by Karl Fischer (KF) analysis. Themoisture content was adjusted by adding WFI to the 33F/DMSO solution toreach a moisture content of 0.2%.

To initiate the activation, 1,1′-carbonyl-di-1,2,4-triazole (CDT) wasfreshly prepared as 100 mg/mL in DMSO solution. Pn33F polysaccharide wasactivated with various amounts of CDT prior to the thiolation step. TheCDT activation was carried out at 23+2° C. for 1 hour. The activationlevel was determined by HPLC (A220/A205). Sodium tetraborate, 100 mM, pH9.0 solution was added to quench any residual CDT in the activationreaction solution. Calculations are performed to determine the addedamount of tetraborate and to allow the final moisture content to be 1.2%of total aqueous. The reaction was allowed to proceed for 1 hour at23±2° C.

Thiolation of Activated Pn-33F Polysaccharide

Cystamine-dihydrochloride was freshly prepared in anhydrous DMSO and 1mol. eq. of cystamine dihydrochloride was added to the activatedpolysaccharide reaction solution. The reaction was allowed to proceedfor 21±3 hours at 23±2° C. The thiolated saccharide solution was diluted10-fold by addition to pre-chilled 5 mM sodium succinate in 0.9% saline,pH 6.0. The diluted reaction solution was filtered through a 5 μmfilter. Dialfiltration of thiolated Pn-33F polysaccharide was carriedout with 100K MWCO ultrafilter membrane cassettes, using Water forInjection (WFI).

Reduction and Purification of Activated Thiolated Pn-33F Polysaccharide

To the retentate a solution of tris(2-carboxyethyl)phosphine (TCEP), 5mol. eq., was added after dilution by 10% volume of 0.1M sodiumphosphate buffer, pH 6.0. This reduction reaction was allowed to proceedfor 2±1 hours at 23±2° C. Dialfiltration of thiolated 33F polysaccharidewas carried out with 100K MWCO ultrafilter membrane cassettes.Diafiltration was performed against pre-chilled 10 mM sodium phosphate,pH 4.3. The thiolated 33F polysaccharide retentate was pulled for bothsaccharide concentration and thiol (Ellman) assays.

Alternative Reduction and Purification of Activated Thiolated Pn-33FPolysaccharide

As an alternative to the purification procedure described above, 33Factivated thiolated saccharide was also purified as follows.

To the thiolated saccharide reaction mixture a solution oftris(2-carboxyethyl)phosphine (TCEP), 5 mol. eq., was added and allowedto proceed for 3±1 hours at 23±2° C. The reaction mixture was thendiluted 5-fold by addition to pre-chilled 5 mM sodium succinate in 0.9%saline, pH 6.0 and filtered through a 5 μm filter. Dialfiltration ofthiolated saccharide was performed using 40-fold diavolume ofpre-chilled 10 mM sodium phosphate monobasic, pH 4.3 with 100K MWCOultrafilter membrane cassettes.

The thiolated 33F polysaccharide retentate was pulled for bothsaccharide concentration and thiol (Ellman) assays. A flow diagram ofthe activation process is provided in FIG. 8(A).

Conjugation Process

Conjugation of Thiolated Pn33F Polysaccharide to Bromoacetylated CRM₁₉₇

The CRM₁₉₇ carrier protein was activated separately by bromoacetylation,as described in Example 1, and then reacted with the activated Pn-33Fpolysaccharide for the conjugation reaction. Before starting theconjugation reaction, the reaction vessel was pre-cooled to 5° C.Bromoacetylated CRM₁₉₇ and thiolated 33F polysaccharide were mixedtogether in a reaction vessel at an agitation speed of 150-200 rpm. Thesaccharide/protein input ratio was 0.9±0.1. The reaction pH was adjustedto 8.0-9.0. The conjugation reaction was allowed to proceed at 5° C. for20±2 hours.

Capping of Reactive Groups on Bromoacetylated CRM₁₉₇ and Thiolated Pn33FPolysaccharide

The unreacted bromoacetylated residues on CRM₁₉₇ proteins were capped byreacting with 2 mol. eq. of N-acetyl-L-cysteine for 3 hours at 5° C.,followed by capping any residual free sulfhydryl groups of the thiolated33F-polysaccharide with 4 mol. eq. of iodoacetamide (IAA) for 20 hoursat 5° C.

Purification of eTEC-Linked Pn-33F Glycoconjugate

The conjugation solution was filtered through a 0.45 μm or 5 μm filter.Dialfiltration of the 33F glycoconjugate was carried out with 300K MWCOultrafilter membrane cassettes. Diafiltration was performed against 5 mMsuccinate-0.9% saline, pH 6.0. The Pn-33F glycoconjugate 300K retentatewas then filtered through a 0.22 μm filter and stored at 5° C. A flowdiagram of the conjugation process is provided in FIG. 8(B).

Results

The reaction parameters and characterization data for several batches ofPn-33F eTEC glycoconjugates are shown in Table 1. The CDTactivation-thiolation with cystamine dihydrochloride generatedglycoconjugates having from 63% to 90% saccharide yields and <1% to 13%free saccharides.

TABLE 1 Experimental Parameters and Characterization Data of Pn33F eTECConjugates Conjugate Batch 33F-1A 33F-2B 33F-3C 33F-4D 33F-5E 33F-6F33F-7G Activation level (mol of 0.21 0.13 0.164 0.103 0.183 0.22 0.19thiol/mol of polysaccharide) Activation level 21 13 16.4 10.3 18.3 22 19(% Thiol) Saccharide/Protein 0.75 1.0 0.75 1.0 1.0 0.75 0.80 (Input)ratio Saccharide yield (%)   69%  63%  71%  63%  69% 82% 90%Saccharide/Protein 1.3 1.7 1.2 1.9 1.6 1.1 1.5 Ratio Free Saccharide12.9% 7.7% 4.4% 7.2% 7.3% <4% <4% MW by SEC-MALLS 2627 2561 4351 29813227 3719 5527 (kDa) CMCA/CMC 14.4/0 13.4/0 6.8/1.9 2.7/0.6 5.9/0.68.2/0 11.4/0.6 % K_(d) (≤0.3)  N/A  85%  88%  75%  68% 67% 76%Acetylation level (mol 0.89 1.16 0.99 0.85 0.81 0.85 1.01 of acetate/molof polysaccharide) N/A = not availableOPA Titers of Pn-33F eTEC Glycoconjugates to CRM₁₉₇

Pn-33F OPA titers in mice were determined under standard conditions(similar to the OPA procedures described below for 10A and 22Fconjugates). OPA titers (GMT with 95% CI) at four and seven weeks areshown in Table 2, demonstrating that the serotype 33F Pn glycoconjugateelicited OPA titers in a murine immunogenicity model.

TABLE 2 Pn-33F OPA Titers (GMT with 95% Cl) 33F Pn Conjugate 0.001 μg0.01 μg 0.1 μg week 4 4 (4, 5)  37 (17, 82) 414 (234, 734)  week 7 8 (5,13) 131 (54, 314) 17567 (9469, 32593)

Example 3. Preparation of Additional Pn-33F eTEC Conjugates

Additional Pn-33F eTEC Conjugates were generated using the processdescribed in Example 2. The reaction parameters and characterizationdata for these additional batches of Pn-33F eTEC glycoconjugates areshown in Table 3.

TABLE 3 Experimental Parameters and Characterization Data of furtherPn33F eTEC Conjugates 33F- 33F- 33F- 33F- 33F- 33F- 33F- 33F- 33F-Conjugate Batch 8H 9I 10J 11K 12L 13M 14N 15O 16P Activation level 0.220.11 0.11 0.13 0.14 0.13 0.06 0.13 0.11 (mol of thiol/mol ofpolysaccharide) Saccharide/Protein (Input) ratio 0.75 0.8 0.8 0.8 0.80.8 0.8 0.8 0.8 Saccharide yield (%) 78%  88%  89%  67%  69% 86%  81% 91%  88% Saccharide/Protein Ratio 1.0 2.2 2.1 1.4 1.4 1.4 2.2 1.9 1.9Free Saccharide <1% 6.8% 5.9% 2.3% 3.6% LOQ 8.2% 3.6% 6.6% MW bySEC-MALLS (kDa) 4729 3293 3295 2246 2498 5539 3070 6009 3789 CMCA/CMC6.6/LOQ 14.2/2.1 15.4/2.1 5.5/1 5.4/1.1 NA/LOQ 1.7/1.2 4.1/2.2 2.2/1.2 %K_(d) (≤0.3) 69% N/A N/A N/A N/A 88%  87%  87%  85% Acetylation level(mol of acetate/mol 0.86 0.93 0.87 1.01 0.99 0.71 0.78 0.8 0.82 ofpolysaccharide) LOQ = limit of quantitation; N/A = not available

As shown above and in Table 3, several Pn-33F conjugates were obtainedusing the eTEC conjugation above. The eTEC chemistry allowed preparationof conjugates with high yield, low % free saccharide and high degree ofconjugation (conjugated lysines). Additionally, it was possible topreserve more than 80% of acetyl functionality using the eTECconjugation process.

Example 4. Evaluation of Pn-33F eTEC Glycoconjugates Stability: % FreeSaccharide Trends

Aliquots of conjugate batch 33F-2B (see table 1) were dispensed intopolypropylene tubes and stored at 4° C., 25° C., and 37° C.,respectively and monitored for trends in % free saccharide. The data (%free saccharide) are shown in Table 4. As shown in this Table, therewere no significant changes in the % free saccharide.

TABLE 4 % Free Saccharide Stability for Pn-33F eTEC Glycoconjugate at 4°C., 25° C. and 37° C. Free Saccharide (%) Lot# Time 33F-2B 0 1 wk 3 wks1 M 2 M 3 M 6 M 4° C. 7.7 N/A 8.3 N/A 9.7 11.2 13 25° C. 7.7 N/A 10.8N/A 11.8 N/A N/A 37° C. 7.7 12.1 N/A 13.4 N/A N/A N/A wk = week; M =month; N/A = not available.

The accelerated stability of another conjugate lot (Batch 33F-3C) wasalso conducted at 37° C. up to 1 month. As shown in Table 5, there wasno significant change to % free saccharide at 37° C., up to 1 month.

TABLE 5 % Free Saccharide Stability for Pn- 33F eTEC Glycoconjugate at37° C. Free Saccharide (%) Time 0 1 day 1 wk 2 wks 1 M Lot# 37° C.33F-3C 4.4 5.9 6.4 7.1 7.2

To further confirm the stability of eTEC conjugates, additionalconjugate batches (33F-3C and 33F-5E (see Table 1)) stored at 4° C. weremonitored up to approximately one year, for potential trends in % freesaccharide. As shown in Table 6, there were no significant changes in %free saccharide levels for the conjugates stored at 4° C. for anextended period up to approximately one year.

TABLE 6 % Free Saccharide Stability Results for Pn-33F eTECGlycoconjugates at 4° C. Free Saccharide (%) Time 0 3 M 4 M 12 M 14 MLot# 4° C. 33F-3C 4.4 N/A 5.3 N/A 7.6 33F-5E 7.3 6.3 N/A 7.4 N/A M =month; N/A = not available

The Serotype 33F conjugates generated by 33F eTEC chemistry weredemonstrated to be stable without noticeable degradation as monitored bythe free saccharide trends at various temperatures (real time andaccelerated).

Example 5. Preparation of Pn-8 Conjugates to CRM₁₉₇ Preparation of Pn-8RAC/DMSO Glycoconjugates

Frozen polysaccharide was thawed and transferred to the reaction vessel.2M acetic acid and WFI (Water for Injection) was added to thepolysaccharide solution to achieve a final polysaccharide concentrationof about 2.5 g/L and a final acetic acid concentration of 0.2M.

Hydrolysis of the Polysaccharide

The native polysaccharide was chemically hydrolyzed prior to activation.The diluted polysaccharide solution was heated to 70° C., and then heldthis temperature for 3.5 hours.

Oxidation of the Polysaccharide

Oxidation of polysaccharide was initiated by the addition of sodiumperiodate solution and the reaction kept to proceed for 20 hrs at 23° C.

Purification of Activated Polysaccharide

The activated polysaccharide was concentrated using ultrafiltrationcassettes. Diafiltration was performed against 20-fold diavolume of WFI.

Lyophilization

The activated polysaccharide is compounded with sucrose to a ratio of 25grams of sucrose per gram of activated polysaccharide. The bottlescontaining the activated saccharide and sucrose are shell frozen inethanol baths and lyophilized.

Conjugation of Activated Polysaccharide to CRM₁₉₇ and Capping

Lyophilized activated polysaccharide was reconstituted to 2 mg/mL inDMSO. DMSO was added to lyophilized CRM₁₉₇ for reconstitution.Reconstituted CRM₁₉₇ was added to the reconstituted activatedpolysaccharide. Conjugation was then initiated by adding sodiumcyanoborohydride to the reaction mixture and was incubated at 23° C. for24 hrs. Termination of conjugation reaction is done by adding 2MEq ofsodium borohydride. This capping reaction proceeded for 3 hrs at 23° C.

Purification of Conjugate

The conjugate solution was then diluted into chilled 5 mM succinate-0.9%saline (pH 6.0), filtered, concentrated to 2-4 g/L using 300K cellulosemembranes, and a first-stage diafiltration was performed against 5 mMsuccinate-0.9% saline (pH6.0). A final purification step was done bydiafiltration with 5 mM succinate-0.9% saline, pH 6.0 buffer. After thediafiltration is completed, the purified conjugate was transferred to acollection tank through a 0.22 μm filter.

Dilution of the Monovalent Bulk Conjugate

The conjugate was diluted further with 5 mM succinate/0.9% saline (pH6), to a target saccharide concentration of 0.5 mg/mL. Final 0.22 μmfiltration step was completed to prepare the monovalent bulk conjugate(MBC) product for formulation.

Several conjugates were obtained using the above described process byvarying different parameters (e.g., saccharide-protein input ratio,reaction concentration and Meq of sodium cyanoborohydride).Characterization for representative Pn-8 glycoconjugates to CRM₁₉₇ isprovided in Table 7.

TABLE 7 Characterization of Pn8-CRM₁₉₇ Conjugates Sample No. 1 2 3 4 5 67 8 9 Activated Saccharide 267 270 352 65 233 340 113 250 230 MW byMALLS (kDa) Saccaride/Protein 0.81 0.84 0.5 2.7 1.15 1.0 0.81 0.64 0.42Ratio MW by SEC-MALLS 12200 8670 3460 3379 4748 4255 5470 9924 6787(kDa)

The Opsonophagocytic activity (OPA) titers for Serotype 8-CRM₁₉₇conjugates in mice were determined in mice under standard conditions(similar to the OPA procedures described below for 10A and 22Fconjugates). OPA titers (geometric mean titer (GMT) with 95% confidenceinterval (CI)) at four weeks at different doses are shown in Table 8 and9 (two separate experiments), demonstrating that the serotype 8conjugate (Samples 1-9; also see Table 7 for characterization data ofthese conjugates) elicited OPA titers in a murine immunogenicity model.

As shown in Table 8, serotype 8 conjugates were shown to havesignificantly higher antibody titers, compared to the controlunconjugated polysaccharide which had poor antibody titers.

TABLE 8 Immunogenicity of Serotype 8-CRM₁₉₇ Conjugates OPA GMT (95% CI)Sample No. 0.001 μg 0.01 μg 0.1 μg 1 17 (10, 30) 88 (47, 165) 1344 (896,2016) 2 7 (4, 11) 184 (87, 387) 1934 (1313, 2847) 3 4 (4, 4) 17 (9, 30)779 (345, 1757) 4 5 (4, 7) 74 (41, 136) 558 (311, 1001) Unconjugated PS13 (3, 55)

TABLE 9 Immunogenicity of Serotype 8-CRM₁₉₇ Conjugates OPA GMT (95% CI)Sample No. 0.001 μg 0.01 μg 5 8 (5, 12) 322 (208, 498) 6 12 (8, 19) 264(129, 537) 7 12 (7, 21) 521 (366, 743) 8 19 (10, 38) 404 (238, 687) 9 33(14, 80) 686 (380, 1237) 2 13 (7, 23) 177 (94, 336)

The overall data generated from conjugates prepared by the abovereductive amination process demonstrated that it allowed preparingconjugates with good conjugation yield, low % free saccharide and withgood stability. Additionally, the prepared conjugates elicited good OPAtiters in a murine immunogenicity model.

Example 6. Preparation of Serotype 10A Polysaccharide—CRM₁₉₇ Conjugate

Preparation of Isolated S. pneumoniae Serotype 10A Polysaccharide

Serotype 10A capsular polysaccharides can be obtained directly frombacteria using isolation procedures known to one of ordinary skill inthe art (see for example methods disclosed in U.S. Patent App. Pub. Nos.2006/0228380, 2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340,and 2008/0102498 and WO 2008/118752). Streptococcus pneumoniae serotype10A were grown in a seed bottle and then transferred to a seedfermentor. Once the targeted optical density was reached, the cells weretransferred to a production fermentor. The fermentation broth wasinactivated by the addition of N-lauroyl sarcosine and purified byultrafiltration and diafiltration.

Oxidation of Isolated Streptococcus pneumoniae Serotype 10A CapsularPolysaccharide

A calculated volume of 0.1M potassium phosphate buffer (pH 6.0) andwater-for-injection (WFI) was added to the polysaccharide solution toachieve a final polysaccharide concentration of 2.5 g/L and a finalconcentration of 25 mM potassium phosphate buffer, if required pH wasadjusted to 6.0, approximately. The diluted polysaccharide was thencooled to 5° C. Oxidation was initiated by the addition of 0.25 molarequivalents (MEq) of sodium periodate solution. The oxidation reactiontime was approximately 4 hrs at 5° C. The oxidation reaction wasquenched with 1MEq of 2,3-butanediol under continuous stirring at 5° C.for 1-2 hrs.

After reaching the target reaction time, the activated polysaccharidewas concentrated using 30K MWCO Millipore ultrafiltration cassettes. Thediafiltration was then performed against 20-fold diavolume of WFI. Thepurified activated polysaccharide was stored at 5° C. The purifiedactivated saccharide is characterized inter alia by (i) Molecular Weightby SEC-MALLS and (ii) Degree of Oxidation.

Conjugation of Activated S. pneumoniae Serotype 10A Polysaccharide withCRM₁₉₇

The conjugation process consisted of the following steps:

a. Compounding with sucrose excipient, and lyophilization;b. Reconstitution of the lyophilized polysaccharide and CRM₁₉₇;c. Conjugation of activated polysaccharide to CRM₁₉₇ and capping; andd. Purification of the conjugate

a. Compounding with Sucrose

The activated polysaccharide is compounded with sucrose to a ratio of 25g of sucrose per gram of activated polysaccharide. The bottle ofcompounded mixture was then lyophilized. Following lyophilization,bottles containing lyophilized activated polysaccharide were stored at−20° C.

b. Reconstitution of Lyophilized Activated Polysaccharide and CRM₁₉₇Protein

Lyophilized activated polysaccharide was reconstituted in anhydrousdimethyl sulfoxide (DMSO). Upon complete dissolution of polysaccharide,the same amount of DMSO was added to the calculated CRM₁₉₇ forreconstitution.

c. Conjugation of Activated Polysaccharide to CRM₁₉₇ and Capping

Reconstituted CRM₁₉₇ (in DMSO) was added to the reconstituted activatedpolysaccharide in the conjugation reactor. The final polysaccharideconcentration is 1 g/L. Conjugation was performed by adding 1.2MEq ofsodium cyanoborohydride to the reaction mixture. The reaction wasincubated and at 23° C. for 24 hrs. Termination of conjugation reactionis done by adding 2MEq of sodium borohydride. The capping reaction wasincubated at 23° C. for 3 hrs.

Termination of conjugation reaction is done by adding 2MEq of sodiumborohydride. This capping reaction proceeded for 3 hrs at 23° C.

d. Purification of Conjugate

The conjugate solution was then diluted into 5× (by volume) chilled 5 mMsuccinate-0.9% saline (pH 6.0) and a 20× diafiltration was performedusing 5 mM succinate-0.9% saline (pH6.0). After the initialdiafiltration was completed, the conjugate retentate was transferredthrough a 0.22 μm filter. The conjugate was diluted further with 5 mMsuccinate/0.9% saline (pH 6), and after the final 0.22 μm filtrationstep it was stored at 2-8° C.

Several conjugates were obtained using the above described process byvarying different parameters (e.g., saccharide-protein input ratio,reaction concentration and MEq of sodium cyanoborohydride). The abovechemistry allowed to generate serotype 10A conjugates which weredemonstrated to be stable without noticeable degradation as monitored bythe free saccharide trends at various temperatures (real time andaccelerated). Characterization for representative Pn-10A glycoconjugatesto CRM₁₉₇ is provided in Table 10.

TABLE 10 Characterization of Pn-10A-CRM₁₉₇ Conjugates Conjugate No. 1 23 4 5 6 DO 12.2 19.5 5.2 10.3 10.8 10.5 Activated Saccharide 191 240 80170 170 170 MW, kDa Input Ratio 1.0 1.0 1.0 1.1 1.1 1.1 % Yield 56 28.565 82 73 66 % Free Saccharide 6.8 10.0 6.7 6.8 6.4 9.7 Conjugate MW, kDa3838 5810 4630 4034 3463 5540 Saccaride/Protein 0.82 0.88 0.85 1.1 1.21.0 Ratio Lys modification 7.4 3.7 13.1 6.9 6.7 6.1 AAA

The opsonophagocytic activity (OPA) titers for Serotype 10A-CRM₁₉₇conjugates in mice were determined under standard conditions. Groups ofthirty 6-7 week old female Swiss Webster mice were immunized with 0.001μg, 0.01 μg, or 0.1 μg of test conjugates via the subcutaneous route onweek 0. The mice were boosted with the same dose of conjugate on week 3and then bled at week 4. Serotype-specific OPAs were performed on week 4sera samples.

Opsonophagocytic activity (OPA) assays are used to measure functionalantibodies in murine sera specific for S. pneumonia serotype 10A. Testserum is set up in assay reactions that measure the ability of capsularpolysaccharide specific immunoglobulin to opsonize bacteria, triggercomplement deposition, thereby facilitating phagocytosis and killing ofbacteria by phagocytes. The OPA titer is defined as the reciprocaldilution that results in a 50% reduction in bacterial count over controlwells without test serum. The OPA titer is interpolated from the twodilutions that encompass this 50% killing cut-off.

OPA procedures were based on methods described in Hu et al. (2005) ClinDiagn Lab Immunol 12 (2):287-295 with the following modifications. Testserum was serially diluted 2.5-fold and added to microtiter assayplates. Live serotype 10A target bacterial strains were added to thewells and the plates were shaken at 37° C. for 30 minutes.

Differentiated HL-60 cells (phagocytes) and baby rabbit serum (3- to4-week old, PEL-FREEZ®, 12.5% final concentration) were added to thewells, and the plates were shaken at 37° C. for 60 minutes. To terminatethe reaction, 80 μL of 0.9% NaCl was added to all wells, mixed, and a 10μL aliquot were transferred to the wells of MULTISCREEN® HTS HV filterplates (MILLIPORE®) containing 200 μL of water. Liquid was filteredthrough the plates under vacuum, and 150 μL of HYSOY® medium was addedto each well and filtered through. The filter plates were then incubatedat 37° C., 5% CO₂ overnight and were then fixed with Destain Solution(Bio-Rad Laboratories, Inc., Hercules, Calif.). The plates were thenstained with Coomassie Blue and destained once. Colonies were imaged andenumerated on a Cellular Technology Limited (CTL) (Shaker Heights, Ohio)IMMUNOSPOT® Analyzer. Raw colony counts were used to plot kill curvesand calculate OPA titers.

OPA titers (geometric mean titer (GMT) with 95% confidence interval(CI)) at four weeks at different doses are shown in Table 11,demonstrating that the serotype 10A conjugate (Samples 1-3; also seeTable 10 for characterization data of these conjugates) elicited OPAtiters in a murine immunogenicity model. As shown in Table 11, serotype10A conjugates were shown to have significantly higher OPA titers,compared to the control unconjugated polysaccharide, which had a poorOPA response.

TABLE 11 Immunogenicity of Serotype 10A-CRM₁₉₇ Conjugates Sample OPA GMT(95% CI) No. 0.001 μg 0.01 μg 0.1 μg 1 858 (556, 1324) 1015 (610, 1691)4461 (3065, 6494) 2 1411 (737, 2703) 796 (460, 1378) 2873 (1768, 4842) 3322 (180, 574) 1062 (528, 2135) 2618 (1415, 4842) Uncon- 602 (193, 1882)jugated PS

Example 7. Conjugation of Pn Serotype-12F Using TEMPO/NCS

In order to improve the stability of serotype 12F-CRM₁₉₇glycoconjugates, alternate chemistries were explored using2,2,6,6-Tetramethyl-1-piperidinyloxy free radical (TEMPO) andN-Chlorosuccinimide (NCS) as the cooxidant to oxidize primary alcoholsto aldehyde groups. GC/MS analysis showed that the sites of oxidationwere different from that of periodate-mediated oxidation. In the case ofTEMPO-NCS oxidation, the α-D-Glcp and 2-Glcp were oxidized, whereasα-D-Galp was the major site of oxidation when periodate was used (seeFIG. 4). As described in further detail herein, TEMPO was used incatalytic amounts (≤0.1 molar equivalents) and the desired degree ofoxidation (DO) was achieved by varying the amounts of NCS used.Subsequently several conjugates were synthesized and characterized. Ingeneral, the production of Serotype 12F glycoconjugates was carried outin several phases, as follows:

a) Hydrolysis of Serotype 12F polysaccharide to molecular weights 50 kDato 500 kDab) Activation of Serotype 12F polysaccharide with TEMPO/NCS;c) Purification of the activated polysaccharide;d) Conjugation of activated Serotype 12F to CRM₁₉₇ protein; ande) Purification of Serotype 12F—CRM₁₉₇ conjugates.

Hydrolysis and Oxidation of Serotype 12F

The hydrolysis of the polysaccharide was typically performed underacidic conditions with heating to obtain an average molecular weight inthe desired range of 100 kDa to 350 kDa. A typical experiment isdescribed below.

Hydrolysis

The Serotype 12F polysaccharide solution was added to a jacketedreaction vessel. To this, the required volume of 0.30M Acetic acid andwater for injection (WFI) were added to maintain ˜0.1M acetic acidconcentration. The pH of the solution was adjusted to 3.2±0.3 using 1NNaOH or Glacial Acetic acid. The temperature of the reaction mixture wasincreased to 70±5° C. The reaction mixture was stirred at 70±5° C. for90-120 minutes. The reaction mixture was cooled down to 23±2° C. andneutralized (pH 7.0) by adding 1M NaOH solution. The hydrolyzedpolysaccharide was purified by ultrafiltration/diafiltration against WFIusing 30K MWCO membranes. The solution was filtered through a 0.22 μmfilter and stored at 2 to 8° C. until oxidation. The molecular weight ofthe hydrolyzed polysaccharide was analyzed by SEC-MALLS to ensure thatthe molecular weight met the target range of 100 kDa to 350 kDa.

Partial Oxidation

In one experiment, the serotype 12F polysaccharide was mechanicallysized using pressure homogenization using a microfluidiser to reduce themolecular weight to approximately 100 kDa to 500 kDa. The sizedpolysaccharide was added to a reaction vessel at a concentration of 4.0mg/mL and mixed with bicarbonate/carbonate buffer (0.5M NaHCO₃/0.05MNa₂CO₃ buffer, pH 8.6) at a ratio of 1:1 v/v. To the stirred mixture wasadded ≤0.1 mol equivalent of TEMPO. The reaction was started by theaddition of 0.6 to 1.0 mol equivalent of NCS. The reaction mixture wasstirred at room temperature for 2 hours, after which the activatedpolysaccharide was purified by diafiltration, with WFI using a 30Kultrafiltration membrane. The purified polysaccharide was collected andthe degree of oxidation (DO) was determined by quantitative measurementsof aldehyde (using a 3-methyl-2-benothiazolinone hydrazone (MBTH) assay)and polysaccharide (using an anthrone assay).

In another experiment, the serotype 12F polysaccharide was hydrolyzed toreduce the molecular weight to a molecular weight of approximately 100kDa to 500 kDa. The serotype 12F polysaccharide was added to a reactionvessel and mixed with 0.5M NaHCO₃/0.05M Na₂CO₃ buffer (pH 8.6) at aratio of 1:1 v/v. To the stirred mixture was added 0.6 to 1.0 molarequivalents of NCS dissolved in WFI. The activation was initiated by theaddition of approximately 0.1 molar equivalents of TEMPO dissolved inWFI. The reaction mixture was stirred at room temperature for 2 hours,after which the activated polysaccharide was purified by diafiltrationwith WFI using a 30K ultrafiltration membrane. The purified activatedpolysaccharide was filtered through a 0.2 μm filter and stored at 4° C.before use.

The TEMPO/NCS mediated oxidations were also performed successfully insodium phosphate buffers of pH 6.5, 7.0, 7.5 and 8.0. In some activationexperiments a primary alcohol such as n-propanol was used to quench thereagents in order to avoid saccharide overoxidation. In another set ofexperiments the chemically hydrolysed polysaccharide was subjected tooxidation directly, without the ultrafiltration/diafiltrationpurification step.

Conjugation of Serotype 12F Oxidized Polysaccharide

In one experiment, the purified oxidized Serotype 12F polysaccharide wasadded to a reaction vessel followed by the addition of 0.5M Sodiumphosphate buffer (pH 6.5) to a final buffer concentration of 0.1M. Tothis solution, previously lyophilized CRM₁₉₇ was added and mixedthoroughly in order to obtain a homogenous solution. The pH was adjustedto 6.8 using diluted HCl or 1N NaOH solution. This was followed by theaddition of 1.5 molar equivalents of NaCNBH₃. The reaction mixture wasstirred for 24 hours at room temperature (23° C.) and for 2.5 days at37° C. The reaction mixture was then diluted with 1×0.9% saline and theunreacted aldehyde groups were “capped” with 2 molar equivalents ofsodium borohydride. The capping reaction time was 3 hours.

In another experiment, the purified activated serotype 12F was added toa reaction vessel followed by the addition of 0.5M sodium phosphatebuffer (pH 6.5) to a final buffer concentration of 0.1M. To thissolution, previously lyophilized CRM₁₉₇ was added and mixed thoroughlyto obtain a homogenous solution. The pH was adjusted to 6.8 usingdiluted HCl or 1N NaOH solution. This was followed by the addition of 3molar equivalents of NaCNBH₃. The reaction mixture was stirred for 24hours at 23° C. and for 48 hrs at 37° C. The reaction mixture was thendiluted with 1×0.9% saline and with stirring, the unreacted aldehydegroups were “capped” with 1 molar equivalent sodium borohydride NaBH₄.The capping reaction time was 3 hours.

In another experiment, the purified activated serotype 12F was added toa reaction vessel and mixed with CRM₁₉₇ solution. The mixture waslyophilized and the powder was dissolved in 0.1M sodium phosphate buffer(pH 6.8) to a final saccharide concentration of 5 mg/mL. If needed thepH was adjusted to 6.8 using diluted HCl or 1N NaOH solution. This wasfollowed by the addition of 3 molar equivalents NaCNBH₃. The reactionmixture was stirred for 24 hours at 23° C. and for 48 hrs at 37° C. Thereaction mixture was then diluted with 1×0.9% saline, the unreactedaldehyde groups were “capped” with 1 molar equivalent sodium borohydrideNaBH₄. The capping reaction time was 3 hours.

Conjugate Purification

The capped reaction mixture was filtered using a 5 μm filter and thenpurified using 100K MWCO ultra filtration membranes. The conjugate wasfirst diafiltered using 10 mM succinate/0.9% saline, pH 6.0 buffer. Thepurified conjugate was then filtered through 0.45/0.22 μm filters toobtain the bulk conjugate.

Degree of Oxidation

Successful oxidation of primary alcohols in the serotype 12Fpolysaccharide was achieved using the TEMPO/NCS system. The hydrolyzedSerotype 12F polysaccharides were oxidized to varying degrees ofoxidation (DO) levels by adjusting the amount of NCS cooxidant. Theeffect on DO by varying amounts of NCS using different polysaccharidebatches and molecular weights is shown in FIG. 9. Typically theoxidation reaction is complete in 2 hours as no significant change in DOwas observed after 2 hours.

Several serotytpe 12F conjugates were generated and characterized usingthe TEMPO/NCS oxidized polysaccharide. The results are summarized inTable 12.

TABLE 12 Pneumococcal Serotype 12F-CRM₁₉₇ conjugates Conjugate Batch12F-84A 12F-97B 12F-147C 12F-171D 12F-177-6E 12F-181F Oxidation Time(hr) 2 2 4 2 2 2 Degree of Oxidation 12.0 6.0 9.6 12.0 11.5 11.5 (DO) %Activated 80 71 70 89 86 86 Saccharide Yield Activated Saccharide 137155 170 190 240 240 MW by MALLS (kDa) Conjugation process Lyo-CRMLyo-CRM Lyo-CRM Lyo-CRM Lyo-CRM Co-Lyo Conjugate Results Saccharideyield (%) 51.6 76.8 53.6 76.3 65.8 40.7 Saccharide/Protein 1.2 0.9 1.01.1 1.4 0.9 Ratio % Free Saccharide 24 10 17 20 23 14 MW by SEC-MALLS2050 3000 3600 1500 2400 2100 (kDa)

Example 8. Immunogenicity of Pn-Serotype 12F-CRM₁₉₇ Conjugates Using theTEMPO/NCS Oxidation Method

The opsonophagocytic activity (OPA) titers for serotype 12F-CRM₁₉₇conjugates in mice were determined in mice under standard conditions.OPA titers (geometric mean titer (GMT) with 95% confidence interval(CI)) at four and seven weeks are shown in Table 13, demonstrating thatthe serotype 12F-CRM₁₉₇ conjugate (Batch 12F-97B; also see Table 12 forcharacterization data of this conjugate) elicited OPA titers in a murineimmunogenicity model. The conjugate generated by the TEMPO-NCS was moreimmunogenic than the control conjugate (171 B) generated from theperiodate oxidation.

TABLE 13 Immunogenicity of Serotype 12F-CRM₁₉₇ Conjugates Dose ConjugateSample 0.001 μg 0.01 μg 0.1 μg Periodate Oxidation (171B) Control 4 16172 TEMPO/NCS Oxidation (12F-97B) 40 417 880

Example 9. Evaluation of Pn-12F Glycoconjugates Stability

Comparison of the stability (at 25° C.) of the conjugates generated byperiodate oxidation vs. TEMPO/NCS oxidation (see FIG. 10) demonstratedthat the conjugate generated by the oxidation of the Pn-12Fpolysaccharides were relatively more stable. As shown in FIG. 10, anincrease in the free saccharide over time was observed for theglycoconjugate generated by the periodate oxidation of the Pn-12Fpolysaccharide at 25° C. In contrast, the glycoconjugate prepared usingthe TEMPO/NCS oxidation of the Pn-12F polysaccharide showed nosignificant trends for the free saccharide under similar conditions.

Example 10. Preparation of Serotype 15B Polysaccharide—CRM₁₉₇ Conjugate

Preparation of Isolated Streptococcus pneumoniae Serotype 15BPolysaccharide

Serotype 15B capsular polysaccharides can be obtained directly frombacteria using isolation procedures known to one of ordinary skill inthe art. The S. pneumoniae serotype 15B were grown in a seed bottle andthen transferred to a seed fermentor. Once the targeted optical densitywas reached, the cells were transferred to a production fermentor. Thefermentation was broth was inactivated by the addition of N-lauroylsarcosine and purified by ultrafiltration and diafiltration.

The purified S. pneumoniae serotype 15B polysaccharide was then sized byhigh pressure homogenization using a PANDA 2K® homogenizer (GEA NiroSoavi, Parma, Italy) to produce the isolated S. pneumoniae serotype 15Bpolysaccharide.

Preferably, the isolated S. pneumoniae serotype 15B capsularpolysaccharide obtained by the above process comprises at least 0.6 mMacetate per mM of serotype 15B capsular polysaccharide and has amolecular weight between 50 kDa and 500 kDa, preferably 150 kDa to 350kDa.

Oxidation of Isolated Streptococcus pneumoniae Serotype 15B Capsularpolysaccharide

Polysaccharide oxidation was carried out in 100 mM potassium phosphatebuffer (pH 6.0) by sequential addition of calculated amount of 500 mMpotassium phosphate buffer (pH 6.0) and WFI to give final polysaccharideconcentration of 2.0 g/L. If required, the reaction pH was adjusted topH 6.0, approximately. After pH adjustment, the reaction temperature wasadjusted to 23° C. Oxidation was initiated by the addition ofapproximately 0.25 molar equivalents of sodium periodate. The oxidationreaction was performed at 23° C. during 16 hrs, approximately.

Concentration and diafiltration of the activated polysaccharide wascarried out using 10K MWCO ultrafiltration cassettes. Diafiltration wasperformed against 20-fold diavolumes of WFI. The purified activatedpolysaccharide was then stored at 5° C. The purified activatedsaccharide was characterized inter alia by (i) saccharide concentrationby colorimetric assay; (ii) aldehyde concentration by colorimetricassay; (iii) Degree of Oxidation (iv) Molecular Weight by SEC-MALLS and(v) presence of O-acetyl and glycerol.

SEC-MALLS is used for the determination of the molecular weight ofpolysaccharides and polysaccharide-protein conjugates. SEC is used toseparate the polysaccharides by hydrodynamic volume. Refractive index(RI) and multi-angle laser light scattering (MALLS) detectors are usedfor the determination of the molecular weight. When light interacts withmatter, it scatters and the amount of scattered light is related to theconcentration, the square of the dn/dc (the specific refractive indexincrements), and the molar mass of the matter. The molecular weightmeasurement is calculated based on the readings from the scattered lightsignal from the MALLS detector and the concentration signal from the RIdetector.

The degree of oxidation (DO=moles of sugar repeat unit/moles ofaldehyde) of the activated polysaccharide was determined as follows:

The moles of sugar repeat unit is determined by various colorimetricmethods, example by using Anthrone method. The polysaccharide is firstbroken down to monosaccharides by the action of sulfuric acid and heat.The Anthrone reagent reacts with the hexoses to form a yellow greencolored complex whose absorbance is read spectrophotometrically at 625nm. Within the range of the assay, the absorbance is directlyproportional to the amount of hexose present.

The moles of aldehyde also are determined simultaneously, using MBTHcolorimetric method. The MBTH assay involves the formation of an azinecompound by reacting aldehyde groups (from a given sample) with a3-methyl-2-benzothiazolone hydrazone (MBTH assay reagent). The excess3-methyl-2-benzothiazolone hydrazone oxidizes to form a reactive cation.The reactive cation and the azine react to form a blue chromophore. Theformed chromophore is then read spectroscopically at 650 nm.

Preferably, the activated S. pneumoniae serotype 15B capsularpolysaccharide obtained by the above process comprises at least 0.6 mMacetate per mM of serotype 15B capsular polysaccharide and has amolecular weight between 50 kDa and 500 kDa, preferably 150 kDa to 350kDa.

Conjugation of Activated S. pneumoniae Serotype 15B CapsularPolysaccharide with CRM₁₉₇

The conjugation process consisted in the following steps:

a) Compounding with sucrose excipient and lyophilization;b) Reconstitution of the lyophilized activated polysaccharide andCRM₁₉₇;c) Conjugation of activated polysaccharide to CRM₁₉₇ and capping; andd) Purification of the conjugate

a) Compounding with Sucrose Excipient, and Lyophilization

The activated polysaccharide was compounded with sucrose to a ratio of25 grams of sucrose per gram of activated polysaccharide. The bottle ofcompounded mixture was then lyophilized. Following lyophilization,bottles containing lyophilized activated polysaccharide were stored at−20° C. Calculated amount of CRM₁₉₇ protein was shell-frozen andlyophilized separately. Lyophilized CRM₁₉₇ was stored at −20° C.

b) Reconstitution of Lyophilized Activated Polysaccharide and CRM₁₉₇Protein

Lyophilized activated polysaccharide was reconstituted in anhydrousdimethyl sulfoxide (DMSO). Upon complete dissolution of polysaccharide,an equal amount of anhydrous DMSO was added to lyophilized CRM₁₉₇ forreconstitution.

c) Conjugation and Capping

Reconstituted activated polysaccharide was combined with reconstitutedCRM₁₉₇ in the reaction vessel (input ratio: 0.8:1), followed by mixingthoroughly to obtain a clear solution before initiating the conjugationwith sodium cyanoborohydride. The final polysaccharide concentration inreaction solution is approximately 1 g/L. Conjugation was initiated byadding 1.0-1.5MEq of sodium cyanoborohydride to the reaction mixture andwas incubated at 23° C. for 40-48 hrs. Conjugation reaction wasterminated by adding 2MEq of sodium borohydride (NaBH₄) to cap unreactedaldehydes. This capping reaction continued at 23° C. for 3 hrs

d) Purification of the Conjugate

The conjugate solution was diluted 1:10 with chilled 5 mM succinate-0.9%saline (pH 6.0) in preparation for purification by tangential flowfiltration using 100-300K MWCO membranes. The diluted conjugate solutionwas passed through a 5 μm filter and diafiltration was performed using 5mM succinate-0.9% saline (pH 6.0) as the medium. After the diafiltrationwas completed, the conjugate retentate was transferred through a 0.22 μmfilter.

The conjugate was diluted further with 5 mM succinate/0.9% saline (pH6), to a target saccharide concentration of approximately 0.5 mg/mL.Final 0.22 μm filtration step was completed to obtain theglycoconjugate.

Preferably, the conjugate obtained by the above process comprises atleast 0.6 mM acetate per mM of serotype 15B capsular polysaccharide, hasa molecular weight between 3,000 kDa and 20,000 kDa and has a degree ofconjugation between 2 and 6.

Example 11. Characterization of Glycoconjugate Comprising S. pneumoniaeSerotype 15B Capsular Polysaccharide Covalently Linked to a CRM₁₉₇

Conjugate 1 was prepared by the process of Example 10. Conjugates 2 and3 were prepared by a similar process using different amount of oxidizingagent. Conjugate 4 was prepared by a similar process except that thepurified serotype 15B capsular polysaccharide was not sized and wasactivated to a lower DO (higher oxidation level) and the conjugation wasperformed in aqueous medium. Conjugate 5 was prepared by a similarprocess except that the purified serotype 15B capsular polysaccharidewas sized by chemical hydrolysis and the conjugation was performed inaqueous medium. Conjugates 6 and 7 were prepared by a similar processexcept that the purified serotype 15B capsular polysaccharide was notsized.

The obtained conjugates were characterized and the results aresummarized in Table 14.

TABLE 14 Streptococcus pneumoniae serotype 15B capsular polysaccharide-CRM₁₉₇ conjugates Conjugate 1 2 3 4 5 6 7 Polysaccharide Sized SizedSized Native Hydrolyzed Native Native O-Acetyl; activated 0.69 0.69 0.691.01 0.66 0.76 N/A Polysaccharide (μmol acetate/μmol poly) Solventmedium DMSO DMSO DMSO Aqueous Aqueous DMSO DMSO Activated Polysaccharide11.4 5.8 9.7 4.8 8.8 5 12 DO Activated Polysaccharide 196 218 235 435270 431 460 MW KDa KDa KDa KDa KDa KDa KDa Yield (%) 87.2 64 63.7 96.278.8 24.2 26.2 Saccharide Protein Ratio 0.68 0.65 0.71 1.22 1.29 0.9 1.5Free Saccharide (%) <5 <5 6.1 18.1 14.2 8.8 18 Conjugate MW, SEC- 61907090 7937 1766 1029 6293 4466 MALLS (kDa) O-Acetylation, Conjugate 0.680.7 0.68 0.61 0.44 0.85 N/A (μmol acetate/μmol poly) <0.3 K_(d) (%), SECN/A 73 N/A N/A 62 N/A N/A Degree of Conj (AAA); 3.7 3.9 4.1 N/A 3.4 N/AN/A Modified Lys % O-Acetyl Retained 99% 100% 99.5% 60% 67% 100% N/A inConjugate N/A = not available

The percentage of free polysaccharide is measured by a procedureutilizing aluminum hydroxide gel to bind protein and covalently boundsaccharide for removal by centrifugation. Samples are mixed withphosphate buffered aluminum hydroxide gel and centrifuged. Boundsaccharide is pelleted with the gel and free saccharide remains in thesupernatant. The resulting supernatant and controls samples arequantitated by appropriate colorimetric assays to determine thepercentage of free saccharide and to confirm sufficient removal ofprotein and recovery of saccharide.

For the amino acid analysis the polysaccharide-protein sample is firsthydrolyzed into its individual components as free amino acids, using 6Nhydrochloric acid (HCl) hydrolysis under vacuum and heat (160° C. for 15minutes). After hydrolysis, the samples are analyzed using Amino AcidAnalyzer. The individual amino acids are separated through ion exchangechromatography using a step gradient of sodium citrate buffer withtemperature and flow rate changes. After separation, the amount of eachamino acid residual is quantitatively determined using a postcolumnninhydrin coupling detection system. In this system, the ninhydrin ismixed with the column eluate in the postcolumn reactor system and themixture passed into the photometer. The reaction of ninhydrin witheluated amino acids yields a purple compound that absorbs maximally at570 nm. This absorbance is a linear response (function) of the amount ofα-amino groups present and this reaction provides quantitativecolorimetric assay for all organic compounds with α-amino groups. In thereaction with imino acids such as proline and hydroxylproline, which donot have free amino group, a bright yellow compound is generated andmonitored at 440 nm. The peak areas for each amino acid are calculatedusing both 570 nm and 440 nm wavelength outputs.

The yield is calculated as follows: (amount of polysaccharide in theconjugate×100)/amount of activated polysaccharide.

Conjugates (4 and 5) generated using an aqueous medium demonstratedsignificant loss in O-acetyl levels. Conjugates generated in DMSOsolvent, using native polysaccharide without MW sizing (6 and 7) did notdemonstrate loss in O-acetyl levels. However, the conjugate yields werevery poor in addition to poor filterability characteristics. Conjugatesgenerated in DMSO using polysaccharides that were sized by high pressurehomogenization (1, 2 and 3) had high yield and better filterabilitycharacteristics with significant preservation of O-acetyl levels. Theseconjugates also had very low levels of free polysaccharides.

Example 12. Opsonophagocytic Activity (OPA) Assay Using Pn-Serotype15B-CRM₁₉₇ Conjugates

The immunogenicity of the S. pneumoniae serotype 15B conjugates of theinvention can be assessed using the OPA assay described below.

Groups of 30 6-7 week old female Swiss Webster mice were immunized with0.001 μg, 0.01 μg, or 0.1 μg of test conjugates via the subcutaneousroute on week 0. The mice were boosted with the same dose of conjugateon week 3 and then bled at week 4. Serotype-specific OPAs were performedon week 4 sera samples.

OPAs are used to measure functional antibodies in murine sera specificfor S. pneumoniae serotype 15B. Test serum is set up in assay reactionsthat measure the ability of capsular polysaccharide specificimmunoglobulin to opsonize bacteria, trigger complement deposition,thereby facilitating phagocytosis and killing of bacteria by phagocytes.The OPA titer is defined as the reciprocal dilution that results in a50% reduction in bacterial count over control wells without test serum.The OPA titer is interpolated from the two dilutions that encompass this50% killing cut-off.

OPA procedures were based on methods described in Hu et al. (2005) ClinDiagn Lab Immunol 12 (2):287-295 with the following modifications. Testserum was serially diluted 2.5-fold and added to microtiter assayplates. Live serotype 15B target bacteria were added to the wells andthe plates were shaken at 37° C. for 30 minutes. Differentiated HL-60cells (phagocytes) and baby rabbit serum (3- to 4-week old, PEL-FREEZ®,6.25% final concentration) were added to the wells, and the plates wereshaken at 37° C. for 45 minutes. To terminate the reaction, 80 μL of0.9% NaCl was added to all wells, mixed, and a 10 μL aliquot weretransferred to the wells of MULTISCREEN® HTS HV filter plates(MILLIPORE®) containing 200 μL of water. Liquid was filtered through theplates under vacuum, and 150 μL of HYSOY® medium was added to each welland filtered through. The filter plates were then incubated at 37° C.,5% CO₂ overnight and were then fixed with Destain Solution (Bio-RadLaboratories, Inc., Hercules, Calif.). The plates were then stained withCoomassie Blue and destained once. Colonies were imaged and enumeratedon a Cellular Technology Limited (CTL) (Shaker Heights, Ohio)IMMUNOSPOT® Analyzer. Raw colony counts were used to plot kill curvesand calculate OPA titers.

The immunogenicity of conjugates 1 and 2 has been tested according tothe above mentioned assay. One additional conjugate and an unconjugatednative S. pneumoniae serotype 15B capsular polysaccharide (unconjugatedPS) were also tested in the same assay:

Conjugate 9 was prepared by conjugation of native (i.e., not sized)serotype 15B capsular polysaccharide to CRM₁₉₇ by reductive amination inaqueous solution.

The results are shown at Table 15.

TABLE 15 OPA Titers of Animal Testing using Serotype 15B-CRM₁₉₇Conjugates OPA GMT (95% CI) 0.001 μg 0.01 μg 0.1 μg Conjugate 1 485(413, 569) 804 (565, 1145) 1563 (1048, 2330) Conjugate 2 556 (438, 707)871 (609, 1247) 1672 (1054, 2651) Conjugate 9 395 (329, 475) 856 (627,1168) 1802 (1108, 2930) Unconjugated — — 698 (466, 1045) PS

As shown in the Table 15 above, conjugates 1 and 2, when administered tomice, generated antibodies capable of opsonizing S. pneumoniae serotype15B, triggering complement deposition, thereby facilitating phagocytosisand killing of bacteria by phagocytes. In addition, despite their lowermolecular weight, they also exhibited similar level of immunogenicity ascompared to conjugate 9 which has not been sized.

Example 13. Preparation of Serotype 22F Polysaccharide—CRM₁₉₇ Conjugate

Preparation of Isolated S. pneumoniae Serotype 22F Polysaccharide

The S. pneumoniae serotype 22F were grown in a seed bottle and thentransferred to a seed fermentor. Once the targeted optical density wasreached, the cells were transferred to a production fermentor. Thefermentation was broth was inactivated by the addition of N-lauroylsarcosine and purified by ultrafiltration and diafiltration.

The purified S. pneumoniae serotype 22F polysaccharide was sized by highpressure homogenization using a PANDA 2K® homogenizer (GEA Niro Soavi,Parma, Italy) to produce the isolated S. pneumoniae serotype 22Fpolysaccharide

Oxidation of Isolated S. pneumoniae Serotype 22F Capsular Polysaccharide

Oxidation of polysaccharide was carried out in 100 mM potassiumphosphate buffer (pH 5.8) obtained by sequential addition of calculatedamount of 500 mM potassium phosphate buffer (pH 5.8) and WFI to givefinal polysaccharide concentration of 2.0 g/L. If required, the reactionpH was adjusted to 5.8, approximately. After pH adjustment, the reactiontemperature was lowered to 5° C. Oxidation was initiated by the additionof 0.10 molar equivalents (MEq) of sodium periodate. The targetoxidation reaction time is 16 hrs at 5° C.

The oxidation reaction was quenched with 2MEq of 2,3-butanediol undercontinuous stirring at 5° C. for 1-2 hrs.

Concentration and diafiltration of the activated polysaccharide wascarried out using 100K MWCO ultrafiltration cassettes. Diafiltration wasperformed against 35-fold diavolume of WFI. The purified activatedpolysaccharide was stored at 5° C. The purified activated saccharide ischaracterized inter alia by (i) Molecular Weight by SEC-MALLS (ii)presence of O-acetyl and (iii) Degree of Oxidation.

SEC-MALLS is used for the determination of the molecular weight ofpolysaccharides and polysaccharide-protein conjugates. SEC is used toseparate the polysaccharides by hydrodynamic volume. Refractive index(RI) and multi-angle laser light scattering (MALLS) detectors are usedfor the determination of the molecular weight. When light interacts withmatter, it scatters and the amount of scattered light is related to theconcentration, the square of the dn/dc (the specific refractive indexincrements), and the molar mass of the matter. The molecular weightmeasurement is calculated based on the readings from the scattered lightsignal from the MALLS detector and the concentration signal from the RIdetector.

The degree of oxidation (DO=moles of sugar repeat unit/moles ofaldehyde) of the activated polysaccharide was determined as follows:

The moles of sugar repeat unit is determined by various colorimetricmethods, for example by using Anthrone method. The polysaccharide isfirst broken down to monosaccharides by the action of sulfuric acid andheat. The Anthrone reagent reacts with the hexoses to form a yellowgreen colored complex whose absorbance is read spectrophotometrically at625 nm. Within the range of the assay, the absorbance is directlyproportional to the amount of hexose present.

The moles of aldehyde also are determined simultaneously, using MBTHcolorimetric method. The MBTH assay involves the formation of an azinecompound by reacting aldehyde groups (from a given sample) with a3-methyl-2-benzothiazolone hydrazone (MBTH assay reagent). The excess3-methyl-2-benzothiazolone hydrazone oxidizes to form a reactive cation.The reactive cation and the azine react to form a blue chromophore. Theformed chromophore is then read spectroscopically at 650 nm.

Conjugation of Activated S. pneumoniae Serotype 22F Polysaccharide withCRM₁₉₇

The conjugation process consisted in the following steps:

a. Compounding with sucrose excipient, and lyophilization;b. Reconstitution of the lyophilized polysaccharide and CRM₁₉₇;c. Conjugation of activated polysaccharide to CRM₁₉₇ and capping; andd. Purification of the conjugate

a. Compounding with Sucrose and Lyophilization

The activated polysaccharide was compounded with sucrose (50% w/v inWFI) to a ratio of 25 grams of sucrose per gram of activatedpolysaccharide. The bottle of compounded mixture was then lyophilized.Following lyophilization, bottles containing lyophilized activatedpolysaccharide were stored at −20° C. Calculated amount of CRM₁₉₇protein (target S/P input ratio=1) was shell frozen and lyophilizedseparately. Lyophilized CRM₁₉₇ was stored at −20° C.

b. Reconstitution of Lyophilized Activated Polysaccharide and CRM₁₉₇Protein

Lyophilized activated polysaccharide was reconstituted in anhydrousdimethyl sulfoxide (DMSO). Upon complete dissolution of polysaccharide,an equal amount of anhydrous DMSO was added to lyophilized CRM₁₉₇ forreconstitution.

c. Conjugation of Activated Polysaccharide to CRM₁₉₇ and Capping

Reconstituted CRM₁₉₇ (in DMSO) was combined in the conjugation reactionvessel with the reconstituted activated polysaccharide. The finalpolysaccharide concentration in reaction solution is 1 g/L. Conjugationwas initiated by adding 1.5MEq of sodium cyanoborohydride to thereaction mixture and the reaction was incubated at 23° C. for 20 hrs.Termination of conjugation reaction is done by adding 2MEq of sodiumborohydride. The capping reaction was incubated at 23° C. for 3 hrs.

d. Purification of Conjugate

The conjugate solution was diluted 1:5 with chilled 5 mM succinate-0.9%saline (pH 6.0) in preparation for purification by tangential flowfiltration using 100K MWCO membranes and a 20× diafiltration wasperformed using 5 mM succinate-0.9% saline (pH6.0) as the medium. Afterthe diafiltration was completed, the conjugate retentate was furtherdiluted, filtered through a 0.22 μm filter and stored at 2-8° C.

Several conjugates were obtained using the above described process byvarying different parameters (e.g., saccharide-protein input ratio,reaction concentration and Meq of sodium cyanoborohydride).Characterization for representative Pn-22F glycoconjugates to CRM₁₉₇ isprovided in Table 16

TABLE 16 Pneumococcal Serotype 22F-CRM₁₉₇ conjugates Batch 1 2 3 4 5 6 78 9 10 Degree of 12.6 19.5 17.2 14.0 12.4 14.9 11.1 14.6 14.4 13.7Oxidation (D.O) Activated 540 697 864 92 866 631 614 639 709 416Saccharide MW by MALLS (kDa) Conjugate Results Saccharide/ 0.75 0.87 20.8 0.8 0.4 1.9 0.8 0.65 1.0 Protein Ratio O—Ac (%) 105 100 N/A N/A N/AN/A N/A N/A N/A N/A % Free Saccharide <5 2 15.5 35 <5 <5 33 <5 <5 8 MWby SEC- 2787 1668 2194 1419 5039 10450 1577 3911 3734 4453 MALLS (kDa)N/A = not available

The % O-Acetyl (preserved) level in the final conjugate was calculatedfrom the ratio of the O-Acetyl content of the conjugate (μmol O-Acetylper μmol of the serotype 22F saccharide repeat unit) relative to theO-Acetyl content of the polysaccharide (μmol O-Acetyl per μmol of theserotype 22F saccharide repeat unit).

The immunogenicity of the conjugates obtained above have been assessedusing the opsonophagocytic assay (OPA) described below.

Groups of thirty 6-7 week old female Swiss Webster mice were immunizedwith 0.001 μg, 0.005 μg or 0.01 μg of test conjugates via thesubcutaneous route on week 0. The mice were boosted with the same doseof conjugate on week 3 and then bled at week 4. Serotype-specific OPAswere performed on week 4 sera samples.

Opsonophagocytic activity (OPA) assays are used to measure functionalantibodies in murine sera specific for S. pneumonia serotype 22F. Testserum is set up in assay reactions that measure the ability of capsularpolysaccharide specific immunoglobulin to opsonize bacteria, triggercomplement deposition, thereby facilitating phagocytosis and killing ofbacteria by phagocytes. The OPA titer is defined as the reciprocaldilution that results in a 50% reduction in bacterial count over controlwells without test serum. The OPA titer is interpolated from the twodilutions that encompass this 50% killing cut-off. OPA procedures werebased on methods described in Hu et al. (2005) Clin Diagn Lab Immunol12(2):287-295 with the following modifications. Test serum was seriallydiluted 2.5-fold and added to microtiter assay plates. Live serotype 22Ftarget bacterial strains were added to the wells and the plates wereshaken at 25° C. for 30 minutes. Differentiated HL-60 cells (phagocytes)and baby rabbit serum (3- to 4-week old, PEL-FREEZ®, 12.5% finalconcentration) were added to the wells, and the plates were shaken at37° C. for 45 minutes. To terminate the reaction, 80 μL of 0.9% NaCl wasadded to all wells, mixed, and a 10 μL aliquot were transferred to thewells of MULTISCREEN® HTS HV filter plates (MILLIPORE®) containing 200μL of water. Liquid was filtered through the plates under vacuum, and150 μL of HYSOY® medium was added to each well and filtered through. Thefilter plates were then incubated at 37° C., 5% CO₂ overnight and werethen fixed with Destain Solution (Bio-Rad Laboratories, Inc., Hercules,Calif.). The plates were then stained with Coomassie Blue and destainedonce. Colonies were imaged and enumerated on a Cellular TechnologyLimited (CTL) (Shaker Heights, Ohio) IMMUNOSPOT® Analyzer. Raw colonycounts were used to plot kill curves and calculate OPA titers.

The Opsonophagocytic activity (OPA) titers for Serotype 22F-CRM₁₉₇conjugates were determined as mentioned above. OPA titers (geometricmean titer (GMT) with 95% confidence interval (CI)) at four weeks atdifferent doses are shown in Tables 17 and 18, (two separateexperiments) demonstrating that the serotype 22F conjugate (Batches 1-7;also see Table 16 for characterization data of these conjugates)elicited OPA titers in a murine immunogenicity model.

TABLE 17 Immunogenicity of Serotype 22F-CRM₁₉₇ Conjugates OPA GMT (95%CI) Sample No. 0.001 μg 0.005 μg 0.01 μg 1 86 (45, 165) 597 (285, 1252)2519 (1409, 4504) 2 98 (51, 191) 782 (410, 1492) 2236 (1319, 3790) 3 35(18, 69) 250 (122, 512) 509 (273, 950)

TABLE 18 Immunogenicity of Serotype 22F-CRM₁₉₇ Conjugates OPA GMT (95%CI) Sample No. 0.001 μg 0.01 μg 4 37 (18, 76) 3383 (1911, 5987) 5 45(20, 103) 1773 (1072, 2931) 6 235 (108, 513) 4335 (3018, 6226) 7 10 (7,13) 252 (138, 457)

Example 14. Preparation of Pn-11A Conjugates to CRM₁₉₇

Preparation of Pn-11A RAC Glycoconjugates

The frozen sized polysaccharide stored in de-ionized water or 25 mMpotassium phosphate buffer (pH 6.0) was thawed at 5° C.

Oxidation of Polysaccharide

Polysaccharide oxidation was carried out in 100 mM potassium phosphatebuffer (pH 6.0) by addition of of 500 mM potassium phosphate buffer (pH6.0) and WFI to give final polysaccharide concentration of 2.0 g/L.Oxidation reaction was carried out at 23° C. Oxidation was initiated bythe addition of sodium periodate. The agitation rate ranges from 100-140rpm.

Purification of Activated 11A Polysaccharide

Concentration and diafiltration of the activated polysaccharide wascarried out using ultrafiltration cassettes. Diafiltration was performedagainst 20-fold diavolume of WFI. After 0.22 μm filtration, the purifiedactivated polysaccharide was stored at 5° C.

Conjugation Process Description

The conjugation process consisted in the following steps:

-   -   a. Shell freezing and lyophilization of CRM₁₉₇ protein;    -   b. Reconstitution of the activated polysaccharide and CRM₁₉₇;    -   c. Conjugation of activated polysaccharide to CRM₁₉₇; and    -   d. Purification and dilution of the conjugate

a. Shell Freezing and Lyophilization of CRM₁₉₇ Protein CRM₁₉₇ proteinwas shell-frozen and lyophilized.

b. Reconstitution of Activated Polysaccharide and CRM₁₉₇ Protein

Activated polysaccharide solution (˜10 g/L) was charged into reactorfollowed by addition of calculated amount 0.5N sodium phosphate buffer(pH 7.2). Under stirring, lyophilized CRM₁₉₇ was added and the reactionmixture was stirred for 2-4 hours in order to reach complete dissolutionof CRM₁₉₇.

c. Conjugation and Capping

Conjugation was initiated by adding cyanoborohydride. The reactionmixture was incubated at 23° C. for 72-96 hrs. Termination ofconjugation reaction was done by adding 0.5×WFI followed by 2MEq ofsodium borohydride. This capping reaction was kept at 23° C. for 3-4hrs.

d. Dilution and Initial Purification of Conjugate

The conjugate solution was diluted 1:5 (reaction volume) with 0.15Nsodium phosphate buffer (pH 8.0) in preparation for purification bytangential flow filtration (TFF). Diluted conjugate was mixed in thedilution vessel and then passed through a 5 μm filter. The filteredconjugate solution was then concentrated down to 1-2 g/L. A two-stepsdiafiltration process was performed. In step one, TFF was carried outusing 30× (diafiltration volume) of 0.15N sodium phosphate buffer (pH8.0) followed by 20× of 5 mM succinate-0.9% NaCl (pH6.0). After theinitial diafiltration was completed, the conjugate retentate wastransferred through a 0.45 μm filter into a collection tank.

Final Diafiltration of Conjugate

The final purification step was a 20× diafiltration with 5 mMsuccinate-0.9% NaCl, pH 6.0 medium using regenerated cellulosemembranes.

Dilution of the Monovalent Bulk Conjugate (MBC)

The conjugate was diluted further with 5 mM succinate/0.9% NaCl, pH 6,to a target saccharide concentration of 0.5 mg/mL. Final 0.22 μmfiltration step was completed to prepare the monovalent bulk conjugate(MBC) product for formulation.

Several conjugates were obtained using the above described process byvarying different parameters (e.g., saccharide-protein input ratio,reaction concentration and Meq of sodium cyanoborohydride).Characterization for representative Pn-11A glycoconjugates to CRM₁₉₇ isprovided in Table 19 (batches 1 to 5).

Preparation of Pn-11A Glycoconjugates Using RAC/DMSO

Oxidized polysaccharide was prepared and purified as described above(see Preparation of Pn-11A RAC Glycoconjugates).

Conjugation via Reductive Amination in DMSO (RAC/DMSO) Conjugation of11A through RAC/DMSO consisted of the following steps:

-   -   a. Compounding with sucrose, shell freezing and lyophilization;    -   b. Reconstitution of the lyophilized polysaccharide and CRM₁₉₇;    -   c. Conjugation of activated polysaccharide to CRM₁₉₇; and    -   d. Purification and dilution of the conjugate.

a. Compounding with Sucrose, Shell Freezing and Lyophilization

The activated polysaccharide prepared from sized polysaccharide wascompounded with sucrose (50% w/v in WFI) to a ratio of 25 grams ofsucrose per gram of activated polysaccharide. The components were mixedthe shell-frozen bottle of compounded mixture was then lyophilized.CRM₁₉₇ protein was shell-frozen and lyophilized separately.

b. Reconstitution of Lyophilized Activated Polysaccharide and CRM₁₉₇Protein

Lyophilized activated polysaccharide was reconstituted in DMSO at 2mg/mL concentration. Upon the complete dissolution of polysaccharide,DMSO was added to lyophilized CRM₁₉₇ for reconstitution

c. Conjugation and Capping

Reconstituted CRM₁₉₇ (in DMSO) was combined in the conjugation reactionvessel with the reconstituted activated polysaccharide. The finalpolysaccharide concentration in reaction solution is 1 g/L. Conjugationwas initiated by adding cyanoborohydride to the reaction mixture and wasincubated at 23° C. for 22 hours. Termination of conjugation reaction isdone by adding 2MEq of sodium borohydride. This capping reaction waskept at 23° C. for 3-4 hrs.

d. Purification and Dilution of the Conjugate

The conjugate solution was purified and diluted using a similar processas described above.

Several conjugates were obtained using the above described process byvarying different parameters (e.g., saccharide-protein input ratio,reaction concentration and Meq of sodium cyanoborohydride).Characterization for representative Pn-11A glycoconjugates to CRM₁₉₇obtained by the above process is provided at Table 19 (batches 6 to 8).

TABLE 19 Pneumococcal Serotype 11A-CRM₁₉₇ conjugates Batch 1 2 3 4 5 6 78 Activated Saccharide 207 129 103 199 183 232 113 113 MW by MALLS (kDa)Conjugate Results Saccharide/Protein Ratio 1.24 1.09 1.32 1.47 1.31 10.78 0.68 Acetate (mol/mol PS) 2.72 2.89 2.72 3.2 3.13 N/A N/A N/AGlycerol (mol/mol PS)* 0.62 0.68 0.75 0.51 0.41 N/A N/A N/A MW bySEC-MALLS (kDa) 3224 837 623 827 994 12200 6543 15730 N/A = notavailable *Glycerol was quantitated by High Performance Anion ExchangeChromatography with Pulsed Amperometric Detection (HPAEC-PAD) after itsrelease from the polysaccharide by hydrofluoric acid (HF).

The overall data generated from conjugates prepared by the abovereductive amination processes demonstrated that it allowed preparingconjugates with good conjugation yield, low % free saccharide and withgood stability.

The immunogenicity of the conjugates obtained above have been assessedusing the opsonophagocytic assay (OPA) described below.

Groups of thirty 6-7 week old female Swiss Webster mice were immunizedwith 0.001 μg, 0.005 μg, 0.01 μg, or 0.1 μg of test conjugates via thesubcutaneous route on week 0. The mice were boosted with the same doseof conjugate on week 3 and then bled at week 4. Serotype-specific OPAswere performed on week 4 sera samples.

Opsonophagocytic activity (OPA) assays are used to measure functionalantibodies in murine sera specific for S. pneumonia serotype 11A. Testserum is set up in assay reactions that measure the ability of capsularpolysaccharide specific immunoglobulin to opsonize bacteria, triggercomplement deposition, thereby facilitating phagocytosis and killing ofbacteria by phagocytes. The OPA titer is defined as the reciprocaldilution that results in a 50% reduction in bacterial count over controlwells without test serum. The OPA titer is interpolated from the twodilutions that encompass this 50% killing cut-off. OPA procedures werebased on methods described in Hu et al. (2005) Clin Diagn Lab Immunol 12(2):287-295 with the following modifications. Test serum was seriallydiluted 2.5-fold and added to microtiter assay plates. Live serotype 22Ftarget bacterial strains were added to the wells and the plates wereshaken at 25° C. for 30 minutes. Differentiated HL-60 cells (phagocytes)and baby rabbit serum (3- to 4-week old, PEL-FREEZ®, 12.5% finalconcentration) were added to the wells, and the plates were shaken at37° C. for 60 minutes. To terminate the reaction, 80 μL of 0.9% NaCl wasadded to all wells, mixed, and a 10 μL aliquot were transferred to thewells of MULTISCREEN® HTS HV filter plates (MILLIPORE®) containing 200μL of water. Liquid was filtered through the plates under vacuum, and150 μL of HYSOY® medium was added to each well and filtered through. Thefilter plates were then incubated at 37° C., 5% CO₂ overnight and werethen fixed with Destain Solution (Bio-Rad Laboratories, Inc., Hercules,Calif.). The plates were then stained with Coomassie Blue and destainedonce. Colonies were imaged and enumerated on a Cellular TechnologyLimited (CTL) (Shaker Heights, Ohio) IMMUNOSPOT® Analyzer. Raw colonycounts were used to plot kill curves and calculate OPA titers.

The Opsonophagocytic activity (OPA) titers for serotype 11A-CRM₁₉₇conjugates in mice were determined as mentioned above. OPA titers(geometric mean titer (GMT) with 95% confidence interval (CI)) at fourweeks at different doses are shown in Table 20, demonstrating that theserotype 11A conjugate (Batches 2-4 and 8; also see Table 19 forcharacterization data of these conjugates) elicited OPA titers in amurine immunogenicity model.

TABLE 20 Immunogenicity of Serotype 11A-CRM₁₉₇ Conjugates OPA GMT (95%CI) Batch No. 0.001 μg 0.01 μg 0.1 μg 2 326 (260, 408) 1391 (794, 2437)4366 (3063, 6223) 3 389 (316, 478) 1113 (690, 1795) 5527 (3698, 8260) 4192 (149, 248) 926 (661, 1298) 2800 (1975, 3970) 8 303 (224, 411) 1099(624, 1935) 3861 (2629, 5669)

Example 15. Formulation of a 16-valent Pneumococcal Conjugate Vaccine

A 16-valent conjugates composition comprising glycoconjugates from S.pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F,22F, 23F and 33F (16vPnC) all individually conjugated to CRM₁₉₇ wasformulated.

Glycoconjugates from S. pneumoniae from serotypes 15B, 22F and 33F wereproduced as disclosed above and S. pneumoniae glycoconjugates fromserotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F wereproduced as disclosed in WO 2006/110381.

The required volumes of bulk concentrates were calculated based on thebatch volume and the bulk saccharide concentrations. The formulated bulkvaccine was prepared by adding the required volume of NaCl/succinatebuffer (pH 5.8) to obtain a final target buffer concentration ofsuccinate 5.0 mM and 150 mM NaCl. Polysorbate 80 to a finalconcentration of 0.02% and the 16 pneumococcal conjugates were added.The preparation was filtered through a 0.2 μm Millipore PES membrane,followed by the addition of AlPO4. The formulation was mixed to allowfor binding and to achieve homogeneity.

The formulation was then filled into glass syringes to deliver a dosevolume of 0.5 mL. The final dosage form consisted in 2.2 μg of each ofglycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14,15B, 18C, 19A, 19F, 22F, 23F and 33F individually conjugated to CRM₁₉₇,4.4 μg of glycoconjugate from S. pneumoniae serotype 6B, 5 mM succinatebuffer pH 5.8, 0.02% (w/w) PS80, 150 mM NaCl and 0.25 mg/mL aluminum asAlPO₄ for a dose of 0.5 mL. CRM₁₉₇, content was about 38 μg for a doseof 0.5 mL.

Example 16. Formulation of a 20-Valent Pneumococcal Conjugate Vaccine

A 20 valent conjugates composition comprising glycoconjugates from S.pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14,15B, 18C, 19A, 19F, 22F, 23F and 33F (20vPnC) all individuallyconjugated to CRM₁₉₇ was formulated.

Glycoconjugates from S. pneumoniae from serotypes 8, 10A, 11A, 12F, 15B,22F and 33F were produced as disclosed above and S. pneumoniaeglycoconjugates from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A,19F and 23F were produced as disclosed in WO 2006/110381.

The required volumes of bulk concentrates were calculated based on thebatch volume and the bulk saccharide concentrations. The formulated bulkvaccine was prepared by adding the required volume of NaCl/succinatebuffer (pH 5.8) to obtain a final target buffer concentration ofsuccinate 5.0 mM and 150 mM NaCl. Polysorbate 80 to a finalconcentration of 0.02% and the 20 pneumococcal conjugates are added. Thepreparation was filtered through a 0.2 μm Millipore PES membrane,followed by the addition of AlPO₄. The formulation was mixed well toobtain maximum binding of the conjugates to the aluminum.

The formulation is then filled into glass syringes to deliver a dosevolume of 0.5 mL.

The final dosage form consisted in 2.2 μg of each of glycoconjugatesfrom S. pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F,14, 15B, 18C, 19A, 19F, 22F, 23F and 33F individually conjugated toCRM₁₉₇, 4.4 μg of glycoconjugate from S. pneumoniae serotype 6B, 5 mMsuccinate buffer pH 5.8, 0.02% (w/w) PS80, 150 mM NaCl and 0.25 mg/mLaluminum as AlPO4 for a dose of 0.5 mL. CRM₁₉₇, content was about 46 μgfor a dose of 0.5 mL.

Example 17. Immunogenicity of a 16-Valent Immunogenic Composition

The immunogenicity of the 16-valent immunogenic composition (see Example15) was assessed in Rabbits using multiplexed direct Lumineximmunoassays (dLIAs) to measure serotype-specific IgG concentrations insera and serotype-specific OPAs.

Groups of ten 2.5 kg to 3.5 kg female New Zealand white rabbits wereimmunized with the proposed human clinical dose (2.2 μg of conjugateexcept serotype 6B which was at 4.4 μg; plus 0.1 mg aluminum as AlPO₄)via the intramuscular route on week 0. The rabbits were boosted with thesame dose of conjugate vaccine on week 2 and then bled at week 4.Serotype-specific dLIAs and OPAs were performed on week 0 and week 4sera samples.

To quantify the total polysaccharide binding antibody (IgG) specific toeach pneumococcal polysaccharide (PnPS), rabbit sera were evaluated intwo direct Luminex immunoassays (dLIAs; 13-plex dLIA, PREVNAR 13®serotypes and 7-plex dLIA, additional serotypes). The 13-plex assaymeasures anti-PnPS antibodies specific to the 13 serotypes included inthe 13-valent pneumococcal conjugate (PnC) vaccine (1, 3, 4, 5, 6A, 6B,7F, 9V, 14, 18C, 19A, 19F, and 23F) and the 7-plex assay measuresanti-PnPS antibodies to the additional serotypes (15B, 22F, 33F). Eachassay contains a combination of 13 or 7 spectrally distinct magneticmicrospheres coupled to PnPS conjugates (PnPS-PLL conjugates: PnPSconjugated to poly-L-Lysine).

Briefly, reference standard, controls and test sera were firstpre-adsorbed with two Pn absorbents; CWPS1 (cell wall polysaccharidefrom PnA containing C-polysaccharide) and CWPS2 (CWP from acapsular S.pneumoniae serotype 2) to block non-specific antibodies from binding tothe PnPS coating antigen. Following preadsorption, the PnPS-coupledmicrospheres were incubated with appropriately diluted referencestandard serum, controls or rabbit test sera. After incubation, eachmixture was washed and an R-Phycoerythrin-conjugated goat anti-rabbitIgG secondary antibody was added. Fluorescent signals (expressed asmedian fluorescence intensities (MFIs)) were measured using a Bio-Plexreader and correlated to the amount of bound PnPS-specific IgG. Valuesfor test sera are reported as (Units/mL, U/mL).

Serotype-specific OPAs were performed as described above. The OPA titeris the reciprocal of the highest serum dilution resulting in 50%reduction in the number of bacterial colony forming units (CFUs) whencompared to the control without serum (defined as the background CFU).The titer is interpolated from the two dilutions that encompass this 50%killing cut-off.

TABLE 21 16vPnC Total IgG Concentrations and OPA Titers Total IgG (PndLIA) Opsonophagocytic Antibody (OPA) Wk0 Wk4 IgG GMC OPA GMT GMC GMCWk4 95% CI Ratio Wk0 Wk4 Wk4 95% CI Ratio Serotype (μg/ml) (μg/ml)(LCI-UCI) Wk4:Wk0 GMT GMT (LCI-UCI) Wk4:Wk0 1 0.08 28 17-44  369 4 87 55-139 22 3 0.08 88 60-128 1062 4 214 151-304 54 4 0.08 30 14-67  402 4934  551-1583 233 5 0.08 34 18-64  449 4 368 232-584 87 6A 0.03 4615-142 1835 4 3026 1607-5696 756 6B 0.08 89 33-241 1182 4 6156 3043-12453 1539 7F 0.01 50 31-78  3969 6 2917 2013-4227 528 9V 0.03 2415-38  881 5 613 426-883 112 14 0.08 28 20-39  368 19 449 331-610 24 18C0.05 79 45-139 1587 4 1847 1003-3401 462 19A 0.08 120 71-205 1605 4 1410 851-2336 352 19F 0.08 156 96-255 2083 4 3207 1783-5771 802 23F 0.05 3313-84  668 4 997  487-2042 249 15B 0.05 54 40-71  1073 6 741  514-1069116 22F 0.08 158 95-262 2103 5 1078  661-1756 211 33F 0.10 11 6-20 11549 1337  829-2154 27 Abbreviations: GMC, geometric mean concentration;CI, confidence interval; LCI, lower confidence interval; UCI, upperconfidence interval.

Results showed a significant increase in serotype-specific IgG andfunctional OPA antibody responses following two immunizations with16vPnC (Table 21). Serum IgG levels increased more than 2-logs abovebaseline. Similarly, a robust functional OPA antibody response waselicited with a minimum of a 22-fold increase in OPA GMT above baseline.Pre-immune sera (Wk 0) showed undetectable levels of PnPS-specific IgGand functional OPA antibody for the majority of the 16v Pn serotypeswith the exception of serotypes 14 and 33F. Low level OPA titers werepresent for these serotypes but these baseline responses did notadversely affect the antibody response following vaccination.

Example 18. Immunogenicity of a 20-Valent Immunogenic Composition

The immunogenicity of the 20-valent immunogenic composition (as preparedat example 16) was assessed in rabbits using multiplexed direct Lumineximmunoassays (dLIAs) to measure serotype-specific IgG concentrations insera and serotype-specific OPAs.

Groups of ten 2.5 kg to 3.5 kg female New Zealand white rabbits wereimmunized with the proposed human clinical dose (2.2 μg of conjugateexcept serotype 6B which was at 4.4 μg; plus 0.1 mg aluminum as AlPO₄)via the intramuscular route on week 0. The rabbits were boosted with thesame dose of conjugate vaccine on week 2 and then bled at week 4.Serotype-specific dLIAs and OPAs were performed on week 0 and week 4sera samples.

To quantify the total polysaccharide binding antibody (IgG) specific toeach pneumococcal polysaccharide (PnPS), rabbit sera were evaluated intwo direct Luminex immunoassays (dLIAs; 13-plex dLIA, PREVNAR 13®serotypes and 7-plex dLIA, additional serotypes). The 13-plex assaymeasures anti-PnPS antibodies specific to the 13 serotypes included inthe 13-valent pneumococcal conjugate (PnC) vaccine (1, 3, 4, 5, 6A, 6B,7F, 9V, 14, 18C, 19A, 19F, and 23F) and the 7-plex assay measuresanti-PnPS antibodies to the additional serotypes (15B, 22F, 33F). Eachassay contains a combination of 13 or 7 spectrally distinct magneticmicrospheres coupled to PnPS conjugates (PnPS-PLL conjugates: PnPSconjugated to poly-L-Lysine).

Briefly, reference standard, controls and test sera were firstpre-adsorbed with two Pn absorbents; CWPS1 (cell wall polysaccharidefrom PnA containing C-polysaccharide) and CWPS2 (CWP from acapsular S.pneumoniae serotype 2) to block non-specific antibodies from binding tothe PnPS coating antigen. Following preadsorption, the PnPS-coupledmicrospheres were incubated with appropriately diluted referencestandard serum, controls or rabbit test sera. After incubation, eachmixture was washed and an R-Phycoerythrin-conjugated goat anti-rabbitIgG secondary antibody was added. Fluorescent signals (expressed asmedian fluorescence intensities (MFIs)) were measured using a Bio-Plexreader and correlated to the amount of bound PnPS-specific IgG. Valuesfor test sera are reported as (Units/mL, U/mL).

Serotype-specific OPAs were performed as described above. The OPA titeris the reciprocal of the highest serum dilution resulting in 50%reduction in the number of bacterial colony forming units (CFUs) whencompared to the control without serum (defined as the background CFU).The titer is interpolated from the two dilutions that encompass this 50%killing cut-off.

Rabbits immunized with the 20vPnC also demonstrated significantincreases in total IgG and functional OPA antibody titers againstserotypes common to the 16v and 20v formulations as well as to theadditional four serotypes (8, 10A, 11A, and 12F) (Table 22). A 2-logincrease in serum IgG levels across the 20 serotypes was inducedfollowing two immunizations. OPA GMTs elicited with the vaccine were atleast 27-fold above baseline. Low level OPA titers in pre-immune serafor serotypes 14 and 33F were similarly observed following 20vPnCvaccination, but again did not alter the robustness of thepost-vaccination antibody responses.

The 16vPnC and 20vPnC formulations elicited a robust humoral responsethat was both specific for Pneumococcal polysaccharides and associatedwith functional killing of the bacterium (see Tables 21 and 22). Inconclusion, studies shown in Examples 17 and 18 demonstrated goodimmunogenicity of both the 16vPnC and 20vPnC formulations.

TABLE 22 20vPnC Total IgG Concentrations and OPA Titers Total IgG (PndLIA) Opsonophagocytic Antibody (OPA) Wk0 Wk4 IgG GMC OPA GMT GMC GMCWk4 95% CI Ratio Wk0 Wk4 Wk4 95% CI Ratio Serotype (μg/ml) (μg/ml)(LCI-UCI) Wk4:Wk0 GMT GMT (LCI-UCI) Wk4:Wk0 1 0.08 28 19-43 379 4 106 69-164 27 3 0.08 116  76-176 1542 4 286 193-425 72 4 0.08 62 39-97 8214 1477  954-2287 369 5 0.08 49 33-71 648 4 509 350-742 127 6A 0.03 3014-66 1209 4 3682 2743-4944 849 6B 0.08 58 36-94 775 4 4469 3002-66531117 7F 0.02 62  39-101 3681 6 3226 2226-4675 500 9V 0.05 30 19-48 644 6956  634-1442 150 14 0.08 34 20-60 457 12 506 348-736 42 18C 0.05 106 67-166 2115 4 1942 1263-2986 485 19A 0.08 112  73-171 1493 4 15801071-2332 395 19F 0.08 178 119-266 2372 4 3392 2085-5519 848 23F 0.05 48 23-103 960 4 1514  889-2577 378 15B 0.05 70 51-98 1410 6 1332  949-1869210 22F 0.10 172 118-250 1811 5 1304 1000-1700 279 33F 0.12 14 10-20 12054 1490 1117-1989 28 8 0.13 144 100-207 1149 4 1388  988-1949 333 10A0.13 54 31-94 433 5 1129  732-1741 236 11A 0.13 178 125-254 1423 7 10483 6373-17241 1434 12F 0.08 31 15-63 408 4 828  608-1127 191Abbreviations: GMC, geometric mean concentration; CI, confidenceinterval; LCI, lower confidence interval; UCI, upper confidenceinterval.

Example 19. Evaluation of Cross-Reactive Opsonophagocytic ImmuneResponses within Serogroup 9 of Streptococcus pneumoniae

The pneumococcal opsonophagocytic assay (OPA), which measures killing ofS. pneumoniae cells by phagocytic effector cells in the presence offunctional antibody and complement, is considered to be an importantsurrogate for evaluating the effectiveness of pneumococcal vaccines.

Materials and Methods

Two randomly selected subsets of immune sera from adults vaccinated witha 13-valent pneumococcal conjugate vaccine (13v PnC) were tested in OPAassays for the serotypes 9V, 9A, 9L and 9N. The sera were collected fromU.S. clinical trials 6115A1-004 (N=59, post-vaccinated) and 6115A1-3005(N=66, matched pre- and post-vaccination), respectively.

Study 6115A1-3005 (ClinicalTrials.gov Identifier: NCT00546572) was aphase 3, randomized, active-controlled, modified double-blind trialevaluating the safety, tolerability, and immunogenicity of PREVNAR 13®compared with a 23-valent pneumococcal polysaccharide vaccine (23vPS) inambulatory elderly individuals aged 70 years and older who received 1dose of 23vPS at least 5 years before study enrollment (see:http://clinicaltrials.gov/ct2/show/NCT00546572; accessed on Mar. 31,2014).

Study 6115A1-004 (ClinicalTrials.gov Identifier: NCT00427895) was aphase 3, randomized, active-controlled, modified double-blind trialevaluating the safety, tolerability, and immunogenicity of a 13-valentpneumococcal conjugate vaccine (13vPnC) compared to a 23-valentpneumococcal polysaccharide vaccine (23vPS) in adults 60 to 64 years oldwho are naive to 23vPS and the safety, olerability, and immunogenicityof 13vPnC in adults 18 to 59 years old who are naïve to 23vPS (see:http://clinicaltrials.gov/show/NCT00427895; accessed on Mar. 31, 2014).

The 13-valent pneumococcal conjugate vaccine (13vPnC) tested in thesestudies contained conjugates from pneumococcal serotypes 1, 3, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, individually conjugated todiphtheria cross-reacting material 197 (CRM₁₉₇) carrier protein.

OPAs are used to measure functional antibodies in human sera against S.pneumoniae serotypes 9V, 9N, 9A and/or 9L. Test serum is set up in assayreactions that measure the ability of capsular polysaccharide specificimmunoglobulin to opsonize bacteria, trigger complement deposition,thereby facilitating phagocytosis and killing of bacteria by phagocytes.The OPA titer is defined as the reciprocal dilution that results in a50% reduction in bacterial count over control wells without test serum.The OPA titer is interpolated from the two dilutions that encompass this50% killing cut-off.

OPA procedures were based on methods described in Hu et al. (2005) ClinDiagn Lab Immunol 122):287-295. Test heat-inactivated serum was seriallydiluted 2.5-fold and was added together with the target bacteria inassay plates and incubated for 30 minutes with shaking. DifferentiatedHL-60 cells (phagocytes) and baby rabbit serum (3- to 4-week old,PEL-FREEZ®, Arkansas, 12.5% final concentration) were then added to thewells, at an approximate effector to target ratio of 200:1, andincubated at 37° C. with shaking. To terminate the reaction, 80 μL of0.9% NaCl was added to all wells, mixed, and a 10 μL aliquot weretransferred to the wells of MULTISCREEN® HTS HV filter plates(MILLIPORE®) containing 200 μL of water. Liquid was filtered through theplates under vacuum, and 150 μL of HYSOY® medium was added to each welland filtered through. The filter plates were then incubated at 37° C.,5% CO₂ overnight and were then fixed with Destain Solution (Bio-RadLaboratories, Inc., Hercules, Calif.). The plates were then stained withCoomassie Blue and destained once. Colonies were imaged and enumeratedon a Cellular Technology Limited (CTL) (Shaker Heights, Ohio)IMMUNOSPOT® Analyzer.

Statistical Analysis: Pearson two-tailed correlations were calculated.

Results—OPA Responses in 9V, 9A, 9L and 9N

The cross-functional response from immune sera of adults immunized with13vPnC against serotypes 9A, 9L, and 9N, was evaluated in the respectivemicrocolony Opsonophagocytic Assays (mcOPAs), along with the homologousfunctional response to serotype 9V. Two randomly selected subsets ofimmune sera from adults vaccinated with 13vPnC were tested. The serawere collected from U.S. clinical trials 6115A1-004 (N=59,post-vaccinated) and 6115A1-3005 (N=66, matched pre- andpost-vaccination), respectively.

Subjects in study 6115A1-004 were previously naïve to any pneumococcalvaccination and received a single dose of 13vPnC as part of the studyprotocol. The immune sera from study 6115A1-004 shows a similarpercentage of responders for all the serogroups with values of 98.3%,98.3%, 100% and 93.2% for 9V, 9A, 9L and 9N respectively (FIG. 11),supporting the results from 6115A1-3005 (FIG. 12). A relative good OPAtiter correlations were observed between serotypes 9V and 9A (Pearsoncorrelation ρ=0.5456, p<0.0001) or 9L (ρ=0.7353, p<0.0001), but not with9N (ρ=0.1217, p<0.3627).

Subjects in study 6115A1-3005 had previously received 1 dose of 23vPS atleast 5 years before study enrollment and received a single dose of13vPnC as part of the study protocol. Matched pre- and post-vaccinationserum panel (N=66) from adults immunized with 13vPnC (study 6115A1-3005)was evaluated on OPA for the homologous response to serotype 9V and forcross-reactivity of anti-9V antibodies to serotypes 9A, 9L, and 9N. Asshown in FIG. 12, a relatively high immunity (percentage responders) to9V (84%), 9A (66%), 9L (82%) and 9N (86%) was detected in the OPA assaylikely due to their previous immunization with 23vPS, which includesunconjugated polysaccharides from serotypes 9V and 9N. However, thepercentage responders increased to 95% or more for all four serotypesafter vaccination with 13vPnC, which only contains serotype 9V conjugatefrom serogroup 9. The fold-rise in titer values are shown in Table 23and are similar between the serotypes also suggesting cross-reactivity.

TABLE 23 OPA Titer Fold-Rise Matched Pre- and Post-Vaccination, 13vPnCOPA Titers 9V 9A 9L 9N Pre Post Pre Post Pre Post Pre Post GMT 221 132341 308 165 706 322 693 Fold-rise 5.9 7.5 4.2 2.1

A more comprehensive analysis of the OPA titer distribution is shown inthe reverse cumulative distribution curves (RCDC) in FIGS. 13-16. TheRCDCs show an increase in serotype-specific immune response postvaccination for serotypes 9V, 9A, 9L and to a lesser extent 9N. Thecorrelation of the fold-rise of titer of individual matched/samplesbetween 9V 9A, 9V/9L, and 9V/9N were also analyzed using Pearson'scorrelation. Relatively good correlations of fold-rises of titers wereobserved between serotypes 9V and 9A (Pearson correlation ρ=0.8720,p<0.0001) or 9N (ρ=0.5801, p<0.0001), but to a lesser extent with 9L(ρ=0.1804, p<0.1640).

Conclusion

Based on these data, the 13vPnC vaccine is likely to provide broaderserotype coverage by providing additional protection against serotypes9A, 9L, and 9N.

Example 20: Cross-Functional OPA Responses Between Serotype 15B andSerotype 15C

Pneumococcal serogroup 15 includes four structurally-related serotypes:15A, 15B, 15C, and 15F. Serotypes 15B and 15C are undistinguishable bygenetic typing techniques and have similar capsular polysaccharide (PS)composition, except that the 15B-PS is the O-acetylated variant of15C-PS. To understand whether anti-capsular PS antibodies for serotype15B are functionally cross-reacting with serotype 15C, 10 rabbits wereimmunized with 16vPnC (see example 15) and 20vPnC (see example 16)vaccines both containing an immunogenic conjugate comprising S.pneumoniae serotype 15B capsular polysaccharide covalently linked toCRM₁₉₇ as disclosed herein as part of their formulation. Sera from pre-and post-vaccination were tested in OPA assays against serotypes 15B and15C target pneumococcal strains.

Of the 10 rabbits from each group, 100% had OPA response to serotype 15Bfollowing immunization with a serotype 15B conjugate. Of these samesamples, 100% had OPA response to serotype 15C as well (Table 24 andTable 25). Low OPA titers were observed in prevaccination sera in 15COPA. However, over 10-fold GMT OPA titer increase with post vaccinationsera compared to pre vaccination demonstrated that the immunogenicconjugates of the invention induces the formation of antibodies capableof killing serotype 15B and 15C Streptococcus pneumonia in an OPA.

TABLE 24 OPA Titers Against serotypes 15B and 15C strains in Rabbit SeraPre and Post vaccination with 16vPnC 15B OPA 15C OPA Animal wk 0 wk 4 wk0 wk 4 1 4 4129 50 2524 2 4 1645 182 472 3 4 1131 126 818 4 4 3199 501189 5 4 2664 36 727 6 4 4589 68 2492 7 11 3601 169 1137 8 4 1838 165672 9 4 1334 98 528 10 4 1108 204 2425 GMT 4 2222 98 1075

TABLE 25 OPA Titers Against serotypes 15B and 15C strains in Rabbit SeraPre and Post vaccination with 20vPnC 15B OPA 15C OPA Animal wk 0 wk 4 wk0 wk 4 1 4 3784 indeterminable* 2353 2 4 862 480 938 3 4 3056  69 1497 44 1948 indeterminable* 1316 5 4 2360  4 4665 6 4 1594 indeterminable*1835 7 4 4943 172 4085 8 4 2419 117 1458 9 4 1245 indeterminable* 527 104 616 indeterminable* 545 GMT 4 1917  77 1515 *Titer cannot bedetermined due to bad killing curves

Example 21. Formulation of a 7-valent Pneumococcal Conjugate Vaccine

A 7 valent conjugate composition comprising glycoconjugates from S.pneumoniae serotypes 8, 10A, 11A, 12F, 15B, 22F and 33F (7vPnC) allindividually conjugated to CRM₁₉₇ was formulated.

Glycoconjugates from S. pneumoniae from serotypes 8, 10A, 11A, 12F, 15B,22F and 33F were produced as disclosed above.

The required volumes of bulk concentrates were calculated based on thebatch volume and the bulk saccharide concentrations. The formulated bulkvaccine was prepared by adding the required volume of NaCl/succinatebuffer (pH 5.8) to obtain a final target buffer concentration of 5.0 mMsuccinate and 150 mM NaCl. Polysorbate 80 to a final concentration of0.02% and the 7 pneumococcal conjugates are added. The preparation wasfiltered through a 0.2 μm Millipore PES membrane, followed by theaddition of AlPO₄. The formulation was mixed well to obtain maximumbinding of the conjugates to the aluminum.

The formulation is then filled into glass syringes to deliver a dosevolume of 0.5 mL.

The final dosage form consisted of 2.2 μg of each of the glycoconjugatesfrom S. pneumoniae serotypes 8, 10A, 11A, 12F, 15B, 22F and 33Findividually conjugated to CRM₁₉₇, 5.0 mM succinate buffer at pH 5.8,0.02% (w/w) PS80, 150 mM NaCl and 0.25 mg/mL aluminum as AlPO4 for adose of 0.5 mL.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are herebyincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, certain changes and modifications may be practiced withinthe scope of the appended claims.

1.-31. (canceled)
 32. An immunogenic composition comprising aglycoconjugate from S. pneumoniae serotype 15B, a glycoconjugate from S.pneumoniae serotype 22F, a glycoconjugate from S. pneumoniae serotype33F, a glycoconjugate from S. pneumoniae serotype 12F, a glycoconjugatefrom S. pneumoniae serotype 10A, a glycoconjugate from S. pneumoniaeserotype 11A, and a glycoconjugate from S. pneumoniae serotype
 8. 33.The immunogenic composition of claim 32, wherein each of saidglycoconjugates is individually conjugated to CRM₁₉₇.
 34. Theimmunogenic composition of claim 32, wherein said serotype 15Bglycoconjugate has a molecular weight of between 1,000 kDa and 20,000kDa.
 35. The immunogenic composition of claim 32, wherein said serotype15B glycoconjugate has a molecular weight of between 10,000 kDa and16,000 kDa.
 36. The immunogenic composition of claim 32, wherein theratio (w/w) of serotype 15B capsular polysaccharide to CRM₁₉₇ in theserotype 15B glycoconjugate is between 0.5 and
 3. 37. The immunogeniccomposition of claim 32, wherein the ratio (w/w) of serotype 15Bcapsular polysaccharide to CRM₁₉₇ in serotype 15B glycoconjugate isbetween 0.7 and 0.9.
 38. The immunogenic composition of claim 32,wherein said serotype 15B glycoconjugate comprises less than about 50%of free serotype 15B capsular polysaccharide compared to the totalamount of serotype 15B capsular polysaccharide.
 39. The immunogeniccomposition of claim 32, wherein at least 40% of the serotype 15Bglycoconjugates have a K_(D) below or equal to 0.3 in a CL-48 column.40. The immunogenic composition of claim 32, wherein said serotype 15Bglycoconjugate comprises at least 0.1 mM acetate per mM serotype 15Bcapsular polysaccharide.
 41. The immunogenic composition of claim 32,wherein said serotype 15B glycoconjugate comprises at least 0.7 mMacetate per mM serotype 15B capsular polysaccharide.
 42. The immunogeniccomposition of claim 32, wherein the ratio of mM acetate per mM serotype15B capsular polysaccharide in the serotype 15B glycoconjugate to mMacetate per mM serotype 15B capsular polysaccharide in the activatedpolysaccharide is at least 0.6.
 43. The immunogenic composition of claim32, wherein said serotype 15B glycoconjugate comprises at least 0.1 mMglycerol per mM serotype 15B capsular polysaccharide.
 44. Theimmunogenic composition of claim 32, wherein the degree of conjugationof said serotype 15B glycoconjugate is between 2 and
 15. 45. Theimmunogenic composition of claim 32, wherein said serotype 15Bglycoconjugate comprise a saccharide having a molecular weight ofbetween 10 kDa and 1,500 kDa.
 46. The immunogenic composition of claim32, wherein each dose of said immunogenic composition comprises 0.1 μgto 100 μg of polysaccharide of each serotype.
 47. The immunogeniccomposition of claim 32, wherein each dose of said immunogeniccomposition comprises 1.0 μg to 10 μg of polysaccharide of eachserotype.
 48. The immunogenic composition of claim 32, wherein each doseof said immunogenic composition comprises about 1.5 μg to about 3.0 μgof polysaccharide for each glycoconjugate from S. pneumoniae serotype 8,10A, 11A, 12F, 158, 22F, and 33F.
 49. The immunogenic composition ofclaim 32, wherein said immunogenic composition further comprises atleast one antigen selected from the group consisting of a diphtheriatoxoid (D), a tetanus toxoid (T), a pertussis antigen (P), an acellularpertussis antigen (Pa), a hepatitis B virus (HBV) surface antigen(HBsAg), a hepatitis A virus (HAV) antigen, a conjugated Haemophilusinfluenzae type b capsular saccharide (Hib), and inactivated poliovirusvaccine (IPV).
 50. The immunogenic composition of claim 32, wherein saidimmunogenic composition further comprises at least one adjuvant selectedfrom the group consisting of aluminum phosphate, aluminum sulfate oraluminum hydroxide, calcium phosphate, liposomes, an oil-in-wateremulsion, MF59 (4.3% w/v squalene, 0.5% w/v polysorbate 80, 0.5% w/vsorbitan trioleate), a water-in-oil emulsion, MONTANIDE™,poly(D,L-lactide-co-glycolide) (PLG) microparticles andpoly(D,L-lactide-co-glycolide) (PLG) nanoparticles.
 51. The immunogeniccomposition of claim 32, wherein said immunogenic composition furthercomprises at least one adjuvant selected from the group consisting ofaluminum phosphate, aluminum sulfate and aluminum hydroxide.
 52. Theimmunogenic composition of claim 32, wherein said immunogeniccomposition further comprises aluminum phosphate as adjuvant.
 53. Theimmunogenic composition of claim 32, wherein said immunogeniccomposition further comprises aluminum sulfate as adjuvant.
 54. Theimmunogenic composition of claim 32, wherein said immunogeniccomposition further comprises aluminum hydroxide as adjuvant.
 55. Theimmunogenic composition of claim 32, wherein said immunogeniccomposition further comprises a CpG Oligonucleotide.
 56. The immunogeniccomposition of claim 32, wherein said immunogenic composition isformulated in a liquid form.
 57. The immunogenic composition of claim32, wherein said immunogenic composition is formulated in a lyophilizedform.
 58. The immunogenic composition of claim 32, wherein saidimmunogenic composition comprises one or more of a buffer, a salt, adivalent cation, a non-ionic detergent, a cryoprotectant, and ananti-oxidant, or any combination thereof.
 59. The immunogeniccomposition of claim 32, wherein said immunogenic composition comprisesa buffer.
 60. The immunogenic composition of claim 59, wherein saidbuffer has a pKa of about 3.5 to about 7.5.
 61. The immunogeniccomposition of claim 59, wherein said buffer is phosphate, succinate,histidine or citrate.
 62. The immunogenic composition of claim 59,wherein said buffer is succinate at a final concentration of 1.0 mM to10 mM.
 63. The immunogenic composition of claim 32, wherein theimmunogenic composition comprises a salt.
 64. The immunogeniccomposition of claim 63, wherein said salt is selected from the groupconsisting of magnesium chloride, potassium chloride, sodium chloride,and a combination thereof.
 65. The immunogenic composition of claim 32,wherein the immunogenic composition comprises a surfactant, and whereinsaid surfactant is selected from the group consisting of polysorbate 20,polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80,polysorbate 85, Triton N-101, Triton X-100, oxtoxynol 40, nonoxynol-9,triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660hydroxystearate, polyoxyethylene-35-ricinoleate, soy lecithin andpoloxamer.
 66. The immunogenic composition of claim 32, wherein saidimmunogenic composition has a pH of 5.5 to 7.5.